Systems, methods, and devices for self-digitization of samples

ABSTRACT

Systems, methods, and devices for discretizing and analyzing fluidic samples are provided. In one aspect, a microfluidic array for discretizing a fluidic sample comprises one or more flow channels and a plurality of fluidic compartments in fluidic communication with the one or more flow channels. In another aspect, a system for discretizing and analyzing fluidic samples comprises a rotor assembly shaped to receive a microfluidic device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/189,663, filed Jul. 7, 2015, the disclosure of which is hereinincorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under R21 GM103459,awarded by the National Institutes of Health. The U.S. Government hascertain rights in the invention.

BACKGROUND

Discretization of samples into small fluidic compartment-definedvolumes, also referred to herein as “self-digitization,” is valuable inmany chemical and biological applications. Prior mechanisms forself-digitization of samples, however, can be less than ideal in atleast some instances. For instance, nebulizers and agitation-basedemulsion generators may not provide sufficient control for someapplications. Methods based on microfluidic technologies may be lessthan optimal with respect to complexity and cost. In some instances,prior approaches may not be adapted for high-throughput processing andanalysis of large numbers of fluidic samples in parallel.

Thus, there is a need for improved systems, methods, and devices forself-digitization and manipulation of samples. The present disclosureaddresses this need and more.

SUMMARY

The present disclosure provides systems, methods, and devices fordiscretizing and analyzing fluidic samples.

In various aspects, the present disclosure provides a microfluidic arrayfor discretizing a fluidic sample, the array comprising: a proximalarray portion comprising a fluid inlet port and a fluid outlet port; adistal array portion away from the proximal portion, one or more flowchannels each comprising a length extending from the proximal arrayportion to the distal array portion; a proximal end in fluidiccommunication with the fluid inlet port, and a distal end in fluidiccommunication with the fluid outlet port, wherein at least one flowchannel comprises a decreasing cross-sectional dimension along thelength from the proximal array portion to the distal array portion; anda plurality of fluidic compartments in fluidic communication with theone or more flow channels.

In various aspects, the present disclosure provides a microfluidic arrayfor discretizing a fluidic sample, the array comprising: a proximalarray portion comprising a fluid inlet port and a fluid outlet port; adistal array portion away from the proximal portion, one or more flowchannels each comprising a length extending from the proximal arrayportion to the distal array portion; a proximal end in fluidiccommunication with the fluid inlet port, and a distal end in fluidiccommunication with the fluid outlet port, wherein at least one flowchannel comprises an increasing or substantially constantcross-sectional dimension along the length from the proximal arrayportion to the distal array portion; and a plurality of fluidiccompartments in fluidic communication with the one or more flowchannels.

In various aspects, the present disclosure provides a microfluidicdevice for discretizing a fluidic sample, the device comprising: a bodycomprising a proximal body portion and a distal body portion; and aplurality of microfluidic arrays as in any of the embodiments hereinformed in the body such that the one or more flow channels of theplurality of microfluidic arrays extend substantially parallel to eachother from the proximal body portion to the distal body portion.

In various aspects, the present disclosure provides a system fordiscretizing and analyzing fluidic samples, the system comprising: arotor assembly comprising a central axis and a plurality of receptaclesarranged radially around the central axis, each receptacle being shapedto receive a microfluidic device as in any of the embodiments hereinsuch that the proximal body portion of the microfluidic device ispositioned near the central axis and the distal body portion of themicrofluidic device is positioned away from the central axis; a rotaryactuator coupled to the rotor assembly; and one or more processorsconfigured with instructions to cause the system to rotate the rotorassembly around the central axis using the rotary actuator.

In various aspects, the present disclosure provides a method fordiscretizing a fluidic sample, the method comprising providing a deviceas in any of the embodiments herein.

In various aspects, the present disclosure provides a method fordiscretizing a fluidic sample, the method comprising providing a systemas in any of the embodiments herein.

In various aspects, the present disclosure provides a method fordiscretizing and analyzing a fluidic sample, the method comprising:providing a microfluidic device as in any of the embodiments herein;applying a fluidic sample to the fluid inlet ports of the microfluidicdevice, the fluidic sample comprising a plurality of discrete analytes;and rotating the microfluidic device such that the plurality of discreteanalytes are driven into a subset of the plurality of fluidiccompartments of the microfluidic device.

This summary is provided to introduce a selection of concepts insimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a microfluidic device for self-digitization of afluidic sample.

FIG. 2 schematically illustrates a flow channel with a decreasingcross-sectional width.

FIG. 3A illustrates a perspective view of a microfluidic device forself-digitization of fluidic samples.

FIG. 3B illustrates a side view of the device of FIG. 3A.

FIGS. 4A and 4B illustrate microfluidic devices configured for use withmulti-channel pipettes.

FIG. 5 illustrates a rotor assembly of a system for self-digitization offluidic samples.

FIGS. 6A and 6B illustrate first and second configurations of a rotorassembly with removably coupled receptacles, respectively.

FIGS. 7A through 7C illustrate rotor assemblies configured toaccommodate heating and/or cooling of microfluidic devices.

FIG. 8 illustrates a portion of a system for heating and/or cooling ofmicrofluidic devices.

FIG. 9A illustrates an oblique illumination configuration for imaging amicrofluidic device.

FIG. 9B illustrates a direct illumination configuration for imaging amicrofluidic device.

FIG. 10 illustrates a multifunctional system for discretization,heating, and imaging.

FIGS. 11A through 11C illustrate discretizing, heating, and imaging aplurality of fluidic samples using a multifunctional system,respectively.

FIGS. 12A and 12B illustrate a configuration for imaging a microfluidicdevice.

FIGS. 13A through 13D illustrate exemplary imaging results obtained witha camera.

FIG. 14 provides an image of the loading instrument according to certainaspects of the disclosure.

FIG. 15 shows a fluorescence image of a sample-loaded device accordingto certain aspects of the disclosure.

FIG. 16 shows images of a region of the device before (top) and after(bottom) thermalcycling.

DETAILED DESCRIPTION

The present disclosure relates generally to systems, methods, anddevices for self-digitization (also referred to herein as “digitization”or “discretization”) of fluidic samples into small sample volumes. Insome embodiments, a microfluidic device for discretizing a fluidicsample includes a plurality of fluidic compartments, and the fluidicsample is discretized into a plurality of discrete sample volumesdefined by the fluidic compartments. In certain embodiments, themicrofluidic device is configured for centrifugal sample loading, thusimproving ease and flexibility of sample processing. For instance, incertain embodiments, the fluidic compartments are in fluidiccommunication with a tapering flow channel shaped to facilitatesubstantially uniform fluid flow, which provides improved reliabilityand control of the self-digitization procedure. Additionally, someembodiments of the present disclosure enable sample discretization,processing, and analysis to be performed using a single rotor-baseddevice. The approaches presented herein provide high-throughput and lowcost processing of fluidic samples compatible with a wide variety ofanalytical techniques.

Some embodiments of the present disclosure include systems, methods, anddevices for the analysis of species that include, but are not limitedto, chemicals, biochemicals, genetic materials, or biological cells.Potential applications for embodiments of the present disclosure includebut are not limited to: polymerase chain reaction (PCR), digitalpolymerase chain reaction (dPCR), nucleic acid sequence-basedamplification including isothermal amplification (e.g., loop mediatedisothermal amplification (LAMP) and nucleic acid sequence-basedamplification (NASBA)), crystallization of proteins and small molecules,and the analysis of cells (e.g., rare cells or single cells) orbiological particles (e.g., isolated mitochondria) present in biologicalfluids. In some embodiments, the systems, methods, and devices of thepresent disclosure can be used for polymerase chain reaction (PCR),reverse transcriptase PCR (RT-PCR), ligase chain reaction (LCR), loopmediated amplification (LAMP) (RT-LAMP), helicase dependentamplification (HDA) (RT-HDA), recombinase polymerase amplification (RPA)(RT-RPA), and/or strand displacement amplification (SDA) (RT-SDA). Incertain embodiments, the systems, methods, and devices of the presentdisclosure can be used for nucleic acid sequence based amplification(NASBA), transcription mediated amplification (TMA), self-sustainedsequence replication (3SR), and single primer isothermal amplification(SPIA). Other techniques that can be used include, e.g., signal mediatedamplification of RNA technology (SMART), rolling circle amplification(RCA), hyper branched rolling circle amplification (HRCA), exponentialamplification reaction (EXPAR), smart amplification (SmartAmp),isothermal and chimeric primer-initiated amplification of nucleic acids(ICANS), and multiple displacement amplification (MDA).

Microfluidic Arrays for Self-Digitization of Fluidic Samples

In some embodiments of the present disclosure, a microfluidic device forself-digitization of a fluidic sample includes a plurality of fluidiccomponents (e.g., compartments, reservoirs, channels, inlets, outlets,etc.), at least some of which are in fluidic communication with eachother. As used herein, the terms “in fluidic communication with” and“fluidly coupled to” (and variations thereof) refers to the existence ofa fluid path between components, and neither implies nor excludes theexistence of any intermediate structures or components, nor implies thata path is always open or available for fluid flow.

FIG. 1 illustrates a microfluidic device 100 for self-digitization of afluidic sample, in accordance with embodiments. The device 100 includesone or more microfluidic arrays, e.g., a first microfluidic array 102 aand a second microfluidic array 102 b. In certain embodiments, differentmicrofluidic arrays are used to discretize different samples and thusare not in fluidic communication with each other. In some embodiments,the microfluidic array (e.g., microfluidic array 102 a) includes one ormore flow channels 104, a plurality of fluidic compartments 106, one ormore fluid inlet ports 108, and one or more fluid outlet ports 110. Insome embodiments, the one or more flow channels 104 each have a lengthextending from a proximal portion 112 of the array to a distal portion114 of the array. In some embodiments, the arrays of the microfluidicdevice 100 are filled by centrifugal loading, as discussed furtherherein, and the orientation of the array is defined relative to the axisof rotation such that “proximal” refers to the direction towards theaxis of rotation and “distal” refers to the direction away from the axisof rotation. In such embodiments, centrifugation is used to drive fluidfrom the proximal portion 112 of the array to the distal portion 114 ofthe array.

In some embodiments, each flow channel 104 includes a proximal end 116in fluidic communication with the one or more fluid inlet ports 108 anda distal end 118 in fluidic communication with the one or more fluidoutlet ports 110. Optionally, in embodiments where a plurality of flowchannels 104 are used, one or more branching channels 120 are used tocouple the inlet port(s) 108 to the proximal end 116 of each flowchannel 104. In some embodiments, the distal ends 118 of the flowchannels 104 are each coupled to the outlet port(s) 110 via an outletreservoir 122 configured to contain a relatively large volume of fluid(e.g., compared to the sample volumes of the fluidic compartments 106).In certain embodiments, the reservoir 122 is used to contain excessfluid during the filling procedure, as discussed further herein.Similarly, in some embodiments, an inlet reservoir is provided betweenthe inlet port(s) 108 and the proximal ends 116 of the flow channels104.

The fluidic compartments 106 are each coupled to the one or more flowchannels 104 via a respective opening conduit 123. In some embodiments,the fluidic compartments are positioned along the length of acorresponding flow channel 104 between the proximal ends 116 and distalends 118. Accordingly, a fluid sample loaded into the array via theoutlet port(s) 110 is distributed into the fluidic compartments 106 viathe flow channels 104 and is thereby discretized into individual samplevolumes. In some embodiments, centrifugation is used to drive fluid intothe fluidic compartments 106, as discussed further herein. Optionally,each fluidic compartment 106 also includes at least one drainage channel124 coupling the compartment 106 to the flow channel 104. In someembodiments, drainage channels are used to control the filling rate ofthe fluidic compartments 106 and/or the completeness of filling.

In some embodiments, the inlet port(s) 108 and outlet port(s) 110 areboth situated at or near the proximal array portion 112, with thereservoir 122 situated at the distal array portion 114. In suchembodiments, one or more outlet return channels 126 extending from thedistal portion 114 to the proximal portion 112 are used to fluidlycouple the reservoir 122 to the outlet port(s) 108. In alternativeembodiments, the inlet port(s) 108 are situated at the proximal portion112 and the distal port(s) 110 are situated at the distal portion 114,or vice-versa. The arrangement in which both the inlet port(s) 108 andoutlet port(s) 110 are at the proximal portion 112 provides certainadvantages for centrifugal loading. For instance, in some embodiments,having the outlet port(s) 110 at the proximal portion 112 near the axisof rotation causes flow rates to slow as the reservoir 122 fills up, asthe fluid is unable to escape through the outlet(s) 110. This designfeature provides a self-metering mechanism in which a set volume offluid will pass through the fluidic compartment region before flow isautomatically stopped. In various embodiments, this approach alsoreduces the likelihood of inadvertent leakage of the sample from theoutlet port(s) 110 during centrifugation.

The design of the microfluidic arrays described herein can be varied asdesired. For instance, in some embodiments, a microfluidic arrayincludes at least 100, at least 500, at least 1000, at least 5000, atleast 10,000, at least 50,000, at least 100,000, at least 500,000 or atleast 1 million fluidic compartments. In certain embodiments, eachfluidic compartment has a volume of approximately 5 pL, 10 pL, 50 pL,100 pL, 500 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, or 500 nL, or within arange from about 5 pL to about 500 nL. Various types of fluidiccompartments are suitable for use within the embodiments herein. Fluidiccompartments can take on various geometries, including but not limitedto shapes where the cross section is circular, oval, square,rectangular, triangular, or some other polygonal shape, or combinationsthereof. In certain embodiments, a fluidic compartment has rounded orbeveled corners. Some fluidic compartments have symmetrical geometries,while others are asymmetrical in shape.

The fluidic compartments of a microfluidic device can be positioned inmany different orientations, e.g., relative to the flow channel. Incertain embodiments, the fluidic compartments are connected to the topor bottom of the flow channel (“top-harbor” or “bottom-harbor” design).In other embodiments, the fluidic compartments are connected to one ormore sides of the flow channel (“side-harbor” design). In certainembodiments, the “long” axis of the fluidic compartment (the directionof the longest dimension of the fluidic compartment) still runs parallelto the main axis, but the compartment is offset from the channel. Inother embodiments, the long axis of the compartment is perpendicular tothe flow channel. In other embodiments, the “long” axis is positioned atsome other angle relative to the flow axis. A fluidic compartment can besaid to be offset from an axis of flow through the flow channel if aline drawn between the center of compartment and the centerline of theflow channel is longer than the shortest distance between a channel walland its centerline.

In certain embodiments with fluidic compartments on the top/bottom ofthe flow channels, the vertical dimension (e.g., height) is increased.For instance, in some embodiments with fluidic compartments on the sidesof the flow channel, both the vertical and a lateral dimension (e.g.,length or width) are increased. In certain embodiments, the fluidiccompartments are located on just one side of the flow channel. In otherembodiments, the fluidic compartments are located on two sides of theflow channel. Having fluidic compartments on two sides of the flowchannel can apply to both side- and bottom-harbor designs, and incertain embodiments fluidic compartments are positioned on three or foursides.

In some embodiments of the present disclosure, the fluidic compartmentsof a microfluidic array function to discretize samples via geometricdifferences between the fluidic compartments and the flow channelsand/or because of positional differences between the fluidiccompartments and the channels (e.g., the fluidic compartments are offsetfrom the channels). In certain embodiments, one or more of the fluidiccompartment dimensions (e.g., length, width, height, cross-sectionalarea) is greater than a corresponding dimension in the flow channel. Insuch embodiments, the differences between the fluidic compartmentdimensions and the corresponding dimensions of the flow channelfacilitate the expansion of an fluidic sample loaded on the device intothe larger volume of the fluidic compartment. Without being bound bytheory, it is believed that this expansion occurs spontaneously becausethe larger dimensions in the fluidic compartment lowers the interfacialenergy between the two fluids relative to what they are in the flowchannel.

In certain embodiments comprising fluidic compartments above or belowthe flow channels, the vertical dimension of the fluidic compartments,or height, is larger than the height of the channel. For instance, insome embodiments with fluidic compartments on the sides of the flowchannel, both the vertical and a lateral dimension of the fluidiccompartment are larger than the same flow channel dimensions.

In certain embodiments, the space between the downstream end of one ofthe fluidic compartments and the upstream end of a downstream fluidiccompartment is between 0.1 and 3.0 times the length of the fluidiccompartments. In certain embodiments, the space between the downstreamend of one of the fluidic compartments and the upstream end of adownstream fluidic compartment is between 0.1 and 1 times the length ofthe fluidic compartments.

In certain embodiments, the width of the flow channel is greater thanthe width of the fluidic compartments. In certain embodiments, thedifference between the width of the flow channel and the width of thefluidic compartments is between 0.001 and 3 times the width of thefluidic compartments. In certain embodiments, the difference between thewidth of the flow channel and the width of the fluidic compartments isbetween 0.01 and 1.5 times the width of the fluidic compartments.

In certain embodiments, additional constrictive or expansive features inthe flow channel are used to facilitate transport of sample to fluidiccompartments and to discretize samples within the compartments. Forexample, the fluidic compartments and/or the flow channels can bedesigned to have various dimensions according to a desired application.In certain embodiments, the fluidic compartment overlap with thechannel, while in other embodiments, the fluidic compartment are flushwith the channel wall, and in yet other embodiments, the fluidiccompartment are connected to the channel by a protrusion. Alternatively,or in addition to these connections, indents in the channel can ineffect recreate overlap with the channel or the use of a protrusion or aflush meeting of the channel and fluidic compartment, without adjustingthe position of the fluidic compartment relative to the flow axis of thechannel. In certain embodiments, these additional channel features(e.g., indents or protrusions) in the channel are used to redirect flowand/or to help isolate fluidic compartments. The indents and protrusionscan have various shapes and sizes to suite particular performancerequirements. In certain embodiments, such as side-harbor designs, thefeatures are on the same side of the channel as the connection with thefluidic compartment. In some embodiments, constrictive or expansivefeatures are located on the opposite side of the channel. In otherembodiments, there are features on other channel sides as well. Incertain embodiments, such as in bottom-harbor designs, the constrictiveor expansive features are adjacent to the bottom compartments but in theplane of the channel.

The designs of the flow channels of the microfluidic arrays describedherein can be varied as desired. In some embodiments, a microfluidicarray includes at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least16, at least 32, at least 64, at least 128, at least 256, at least 512,at least 1024, at least 2048, at least 4096, or at least 8192 flowchannels. In some embodiments, the flow channels are arranged to extendparallel or substantially parallel to each other. In alternativeembodiments, some or all of the flow channels do not extend parallel toeach other. A flow channel can be linear, curved, or curvilinear, asdesired. In some embodiments, a microfluidic array includes onlyparallel linear flow channels, and such arrays are referred to herein as“linear microfluidic arrays.” A flow channel can have a wide variety ofcross-sectional shapes, such as a rectangular, trapezoidal, circular,semi-circular, oval, semi-oval, square, or triangular cross-sectionalshape. In some embodiments, a flow channel has a single uniformcross-sectional shape throughout the length of the channel, while inother embodiments, the cross-sectional shape is variable along thelength of the channel.

Tapering Flow Channels

In some embodiments, at least one flow channel of a microfluidic arrayhas at least one decreasing cross-sectional dimension (e.g., width,length, height, area) along the length of the channel, e.g., from theproximal to the distal end of the channel and/or from the proximal tothe distal portion of the array. In certain embodiments, “along thelength of the channel” refers to along the portion of the channel thatspans the entirety of the fluidic compartments coupled to the channel.Channels exhibiting a decreasing cross-sectional dimension along thelength of the channel are also referred to herein as “tapering flowchannels.” In some embodiments, only a subset of the flow channels of amicrofluidic array are tapering flow channels, such that at least someof the flow channels of the systems and devices herein do not exhibittapering. In alternative embodiments, all of the flow channels of amicrofluidic array are tapering flow channels, such that each of theflow channels exhibits tapering. The proportion of tapering andnon-tapering channels in a microfluidic array can be varied as desired,e.g., such that approximately 0%, approximately 10%, approximately 20%,approximately 30%, approximately 40%, approximately 50%, approximately60%, approximately 70%, approximately 80%, approximately 90%, orapproximately 100% of the channels of the array exhibit tapering. Insome embodiments, the cross-sectional dimension of at least one flowchannel decreases according to a tapering profile configured to producesubstantially uniform fluid flow rates and/or increasing flow resistancealong the length of the flow channel. In some embodiments, the use offlow channels configured to produce substantially uniform fluid flowand/or increasing flow resistance improves control and consistency ofcompartment filling.

In some embodiments, the tapering profile of the cross-sectionaldimension is determined based on the following relation for centrifugalmicrofluidic systems:

U=(D _(h) ²ρω² rΔr)/(32 μL)

where U is the flow velocity, D_(h) is the hydraulic diameter of thechannel (D_(h) ²=4AP where A is the cross-sectional area of the channeland P is the perimeter of the channel), ρ is the density of the fluid, ωis the angular velocity of the fluid, r is the average distance of thefluid in the channel to the axis of rotation, Δr is the radial extent ofthe fluid, μ is the viscosity of the fluid, and L is the length of thefluid in the channel. The volumetric flow rate Q is related to the flowvelocity U and cross-sectional area A by the relation Q=UA.

In some embodiments, the tapering profile of the cross-sectionaldimension is a continuous tapering profile without discontinuities(e.g., breaks, steps, etc.). For instance, a continuous tapering profilecan be a linear tapering profile that decreases linearly, an exponentialtapering profile that decreases exponentially, a polynomial taperingprofile that decreases according to a polynomial function, orcombinations thereof (e.g., some portions taper linearly while otherportions taper exponentially, etc.). In other embodiments, the taperingprofile of the cross-sectional dimension is a discontinuous taperingprofile, e.g., a stepped tapering profile that decreases according to astep function. Optionally, in certain embodiments, the tapering profileincludes a combination of one or more portions with continuous taperingand one or more portions with discontinuous tapering.

As an example, in some embodiments, some or all of the flow channelspresented herein have a decreasing cross-sectional width along thelength of the channel. In certain embodiments (e.g., when thecross-sectional width varies along the height of the channel), adecrease in cross-sectional width is determined with respect to anaverage cross-sectional width, a maximum cross-sectional width, and/or aminimum cross-sectional width of the flow channel.

FIG. 2 schematically illustrates a flow channel 200 of a microfluidicarray with a decreasing cross-sectional width, in accordance withembodiments. The flow channel 200 is suitable for use with anyembodiment of the systems, methods, and devices presented herein. Theflow channel 200 is coupled to each of a plurality of fluidiccompartments 202 via a respective opening conduit 204 and drainagechannel 206. The fluidic compartments 202 are distributed along thelength of the flow channel 200 between the proximal end 208 and distalend 210 of the flow channel 200. As discussed above and herein, inembodiments where centrifugal filling is used, the proximal end 208 ofthe flow channel 200 is oriented towards the axis of rotation and thedistal end 210 is located away from the center of location, such thatfluid is driven along the length of the flow channel 200 and into thefluidic compartments 202 along a proximal-distal direction. In someembodiments, the flow channel 200 comprises a cross-sectional width 212at or near the proximal end 208 greater than a cross-sectional width ator near the distal end 214. As used herein, “at or near the proximalend” may refer to portions of a flow channel that are proximal to themost proximal fluidic compartment coupled to the channel (e.g.,compartment 216), and “at or near the distal end” may refer to portionsof a flow channel that are distal to the most distal fluidic compartmentcoupled to the channel (e.g., compartment 218).

The dimension of a flow channel exhibiting a tapering cross-sectionalwidth can be varied as desired. For example, in certain embodiments, thecross-sectional width of the channel at or near the proximal end isapproximately 80 μm, or within a range from approximately 60 μm toapproximately 120 μm, and the cross-sectional width of the channel at ornear the distal end is approximately 50 μm, or within a range fromapproximately 30 μm to approximately 70 μm. In various embodiments, thecross-sectional width at or near the proximal end is approximately 1.2times to approximately 2 times greater than the cross-sectional width ator near the distal end, or approximately 1 time to approximately 3 timesgreater than the cross-sectional width near the distal end. In variousembodiments, the cross-sectional width of the flow channel decreases atan average rate within a range from approximately 0.2 μm/mm toapproximately 0.75 μm/mm, from approximately 0.15 μm/mm to approximately0.3 μm/mm, from approximately 0.1 μm/mm to approximately 2 μm/mm, orfrom approximately 0.01 μm/mm to approximately 10 μm/mm along the lengthof the flow channel.

As another example, in some embodiments, some or all of the flowchannels herein have a decreasing cross-sectional area along the lengthof the channel. For example, in certain embodiments, the cross-sectionalarea of the channel at or near the proximal end is approximately 2400μm², or within a range from approximately 1200 μm² to approximately 4800μm², and the cross-sectional area of the channel at or near the distalend is approximately 1500 μm², or within a range from approximately 600μm² to approximately 2800 μm². In certain embodiments, thecross-sectional area of the channel at the proximal end is less than orequal to 10,000 μm². In other embodiments, the cross-sectional area ofthe channel at the distal end is less than or equal to 100 μm².

In alternative embodiments, some or all of the tapering flow channelsherein exhibit an increasing cross-sectional dimension along the lengthof the channel, rather than a decreasing cross-sectional dimension. Insome embodiments, the tapering profile of the cross-sectional dimensionis a continuous tapering profile without discontinuities (e.g., breaks,steps, etc.). For instance, a continuous tapering profile can be alinear tapering profile that increases linearly, an exponential taperingprofile that increases exponentially, a polynomial tapering profile thatincreases according to a polynomial function, or combinations thereof(e.g., some portions taper linearly while other portions taperexponentially, etc.). In other embodiments, the tapering profile of thecross-sectional dimension is a discontinuous tapering profile, e.g., astepped tapering profile that increases according to a step function.Optionally, in certain embodiments, the tapering profile includes acombination of one or more portions with continuous tapering and one ormore portions with discontinuous tapering.

As an example, in some embodiments, some or all of the flow channelspresented herein have a increasing cross-sectional width along thelength of the channel. In certain embodiments (e.g., when thecross-sectional width varies along the height of the channel), anincrease in cross-sectional width is determined with respect to anaverage cross-sectional width, a maximum cross-sectional width, and/or aminimum cross-sectional width of the flow channel.

The dimension of a flow channel exhibiting an increasing cross-sectionalwidth can be varied as desired. For example, in certain embodiments, thecross-sectional width of the channel at or near the distal end isapproximately 80 μm, or within a range from approximately 60 μm toapproximately 120 μm, or within a range from approximately 10 μm toapproximately 200 μm, and the cross-sectional width of the channel at ornear the proximal end is approximately 50 μm, or within a range fromapproximately 30 μm to approximately 70 μm, or within a range fromapproximately 40 μm to approximately 200 μm. In various embodiments, thecross-sectional width at or near the distal end is approximately 1.2times to approximately 2 times greater than the cross-sectional width ator near the proximal end, or approximately 1 time to approximately 3times greater than the cross-sectional width near the proximal end. Invarious embodiments, the cross-sectional width of the flow channelincreases at an average rate within a range from approximately 0.2 μm/mmto approximately 0.75 μm/mm, from approximately 0.15 μm/mm toapproximately 0.3 μm/mm, from approximately 0.1 μm/mm to approximately 2μm/mm, or from approximately 0.01 μm/mm to approximately 10 μm/mm alongthe length of the flow channel.

As another example, in some embodiments, some or all of the flowchannels herein have an increasing cross-sectional area along the lengthof the channel. For example, in certain embodiments, the cross-sectionalarea of the channel at or near the distal end is approximately 2400 μm²,or within a range from approximately 1200 μm² to approximately 4800 μm²,and the cross-sectional area of the channel at or near the proximal endis approximately 1500 μm², or within a range from approximately 600 μm²to approximately 2800 μm².

Constant-Diameter Flow Channels

In some embodiments, one or more flow channels of a microfluidic arrayhave a substantially constant cross-sectional dimension (e.g., width,length, height, area) along the length of the channel, e.g., from theproximal to the distal end of the channel and/or from the proximal tothe distal portion of the array. In certain embodiments, “along thelength of the channel” refers to along the portion of the channel thatspans the entirety of the fluidic compartments coupled to the channel.Channels exhibiting a substantially constant cross-sectional dimensionalong the length of the channel are also referred to herein as“constant-diameter flow channels.” In some embodiments, only a subset ofthe flow channels of a microfluidic array are constant-diameter flowchannels, such that at least some of the flow channels of the systemsand devices herein do not have a substantially constant cross-sectionaldimension along the length of the channel. In alternative embodiments,all of the flow channels of a microfluidic array are constant-diameterflow channels, such that each of the flow channels exhibits asubstantially constant cross-sectional dimension along the length of thechannel. The proportion of constant-diameter and non-constant-diameterchannels in a microfluidic array can be varied as desired, e.g., suchthat approximately 0%, approximately 10%, approximately 20%,approximately 30%, approximately 40%, approximately 50%, approximately60%, approximately 70%, approximately 80%, approximately 90%, orapproximately 100% of the channels of the array exhibit a substantiallyconstant cross-sectional dimension.

In some embodiments, the constant-diameter profile of thecross-sectional dimension is a continuous profile withoutdiscontinuities (e.g., breaks, steps, etc.). For instance, the profilecan be substantially constant across the length of the channel. In otherembodiments, the constant-diameter profile of the cross-sectionaldimension is a continuous profile with discontinuities (e.g., breaks,steps, etc.), but with an average diameter that is substantiallyconstant over the length of the channel.

Optionally, in certain embodiments, the constant-diameter profileincludes a combination of one or more portions without discontinuitiesand one or more portions with discontinuities.

The dimension of a flow channel exhibiting a constant-diametercross-sectional width can be selected as desired. For example, incertain embodiments, the cross-sectional width of the channel isapproximately 80 μm, within a range from approximately 60 μm toapproximately 120 μm, approximately 50 μm, or within a range fromapproximately 30 μm to approximately 70 μm.

In some embodiments, the cross-sectional width of the channel is withina range from approximately 40 μm to approximately 200 μm, or within arange from approximately 10 μm to approximately 200 μm. In someembodiments, the flow channels have cross-sectional areas ofapproximately 2400 μm², within a range from approximately 1200 μm² toapproximately 4800 μm², 1500 μm², within a range from approximately 600μm² to approximately 2800 μm², or within a range from approximately 100μm² to approximately 10,000 μm².

Microfluidic Devices for Self-Digitization of Fluidic Samples

In some embodiments, the present disclosure provides a microfluidicdevice (e.g., a microfluidic chip) having one or more microfluidicarrays for discretizing one or more fluidic samples into a plurality offluidic compartments. In certain embodiments, a microfluidic deviceincludes 2, 4, 6, 8, 12, 16, 24, 32, 36, or 48 microfluidic arraysand/or at least 500, at least 1000, at least 5000, at least 10,000, atleast 50,000, at least 100,000, at least 500,000, at least 1 million, atleast 5 million, or at least 10 million fluidic compartments.

In some embodiments, the microfluidic devices of the present disclosureare configured for centrifugal loading of fluidic samples. In suchembodiments, the microfluidic device includes a device body with aproximal body portion configured to be oriented towards the axis ofrotation during centrifugation, and a distal body portion configured tobe oriented away from the axis of rotation during centrifugation.Accordingly, the one or more microfluidic arrays are formed in the bodyof the microfluidic device with the flow channels (e.g., parallel flowchannels) extending from the proximal body portion to the distal bodyportion, such that centrifugation drives fluid through the arrays fromthe proximal ends of the flow channels to the distal ends of the flowchannels. The plurality of microfluidic arrays can be formed in thedevice body in a variety of ways, such as 3D printing, replica molding,injection molding, embossing, soft lithography, or a combinationthereof.

FIGS. 3A and 3B illustrate a microfluidic device 300 forself-digitization of fluidic samples, in accordance with embodiments.Similar to the device 100 of FIG. 1, the device 300 includes a pluralityof microfluidic arrays each having at least one fluid inlet port 302,one or more flow channels 304, a plurality of fluidic compartments 306,and at least one fluid outlet port 308. In some embodiments, the fluidinlet port(s) 302 are coupled to the flow channels 304 via an optionalinlet reservoir 310 and a branching channel 312, and the fluid outletport(s) 308 are coupled to the flow channels 304 via an optional outletreservoir 314 and outlet return channel 316. Optionally, the fluidiccompartments 306 each include a respective drainage channel 318 coupledto the respective flow channel 304.

In some embodiments, the body of the device 300 is formed from apolydimethylsiloxane (PDMS) layer 320 on a glass slide 322 with aspin-coated PDMS layer 324. The features of the microfluidic arrays(e.g., flow channels 304, fluidic compartments 306) are formed in thePDMS layer 320 in accordance with methods known to one of ordinary skillin the art. In certain embodiments, a first PDMS block 326 is used toform the outlet reservoir 314 and a second PDMS block 328 is used toform the inlet and outlet ports 302, 308 and inlet reservoir 310. Insome embodiments, the first and/or second PDMS blocks 326, 328 areseparate from the PDMS layer 320, while in other embodiments, the firstand/or second PDMS blocks 326, 328 are integrally formed as a singlepiece with the PDMS layer 320. Optionally, a vapor barrier 330 ispositioned over the PDMS layer 320, e.g., to reduce evaporation of fluidthrough the PDMS layer 320.

The depths of the features formed in the device using PDMS can be variedas desired. For instance, in certain embodiments, the fluid inletport(s) 302, fluid outlet port(s) 308, inlet reservoir 310, and/oroutlet reservoir 314 have a depth of at least approximately 0.05 mm, 0.1mm, 0.2 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or 6 mm.

The microfluidic devices of the present disclosure can be designed in avariety of ways. For example, in some embodiments, the body of amicrofluidic device comprises a substantially rectangular shape, asubstantially square shape, a substantially circular shape, asubstantially semi-circular shape, a substantially oval shape, asubstantially semi-oval shape, or any other geometry. In certainembodiments, the body of a microfluidic device is shaped to be similarto existing devices and/or accommodate existing instrumentation, e.g.,for convenience and compatibility. For instance, in various embodiments,the body is substantially rectangular with a length (e.g., along theproximal-distal direction) of approximately 127.8 mm and a width (e.g.,orthogonal to the proximal-distal direction) of approximately 85.5 mm,similar to a 96-well microplate. As another example, in variousembodiments, the body is substantially rectangular with a length ofapproximately 100 mm and a width of approximately 75 mm, similar to theadapter size for certain PCR thermal cycling devices.

In some embodiments, the arrangement of the microfluidic arrays in thebody of the microfluidic device is configured to be compatible withexisting laboratory equipment, such as a multi-channel pipette. Forinstance, some embodiments of the present disclosure providemicrofluidic devices configured for sample loading using a multi-channelpipette. In various embodiments, a multi-channel pipette is used toconcurrently load a plurality of fluidic samples into a plurality offluid inlet ports of a microfluidic device, with each fluid inlet portreceiving a different fluidic sample from a respective channel of thepipette. In certain embodiments, each fluid inlet port is fluidlycoupled to a different microfluidic array, such that the differentfluidic samples can be discretized in parallel in accordance with themethods presented herein. This approach allows for simultaneousdiscretization of multiple samples using a single microfluidic deviceand is advantageous for high-throughput sample processing.

Accordingly, in some embodiments, the fluid inlet ports of themicrofluidic arrays formed in a microfluidic device are arranged toreceive fluidic samples from a multi-channel pipette. In certainembodiments, for example, the fluid inlet ports are arranged in a linearrow (e.g., near the proximal body portion of the device) similar to thearrangement of pipette channels of a multi-channel pipette. In certainembodiments, the number of fluid inlet ports and pitch (e.g.,center-to-center distance) between adjacent fluid ports corresponds tothe number of channels and channel pitch of a multi-channel pipette,respectively. For instance, in various embodiments, a multi-channelpipette includes 4, 6, 8, or 12 pipette channels, with the pitch betweenadjacent pipette channels being approximately 2.25 mm, 3 mm, 4.5 mm, 9mm, 12 mm, 18 mm, or 36 mm. Accordingly, in various embodiments, themicrofluidic device has 4, 6, 8, or 12 fluid inlet ports, with the pitchbetween adjacent ports being approximately 2.25 mm, 3 mm, 4.5 mm, 9 mm,12 mm, 18 mm, or 36 mm.

FIG. 4A illustrates an exemplary microfluidic device 400 configured foruse with multi-channel pipettes, in accordance with embodiments Thedevice 400 includes 8 microfluidic arrays 402 each having a single fluidinlet port 404 for sample loading. Each fluid inlet port 404 is fluidlycoupled to a plurality of flow channels (e.g., 4 flow channels) andfluidic compartments (e.g., 148 fluidic compartments), similar tovarious embodiments presented herein. The 8 fluid inlet ports 404 arearranged in a linear row at the proximal portion 406 of the device 400.In some embodiments, the pitch 408 between adjacent fluid inlet ports isapproximately 9 mm in order to accommodate a standard 8-channel pipette.

FIG. 4B illustrates another exemplary microfluidic device 450 configuredfor use with multi-channel pipettes, in accordance with embodiments.Similar to the device 400, the device 450 includes 8 microfluidic arrays452 each having a single fluid inlet port 454 for sample loading. Eachfluid inlet port 454 is fluidly coupled to a plurality of flow channels(e.g., 16 flow channels) and fluidic compartments (e.g., 2560 fluidiccompartments). The 8 fluid inlet ports 454 are arranged in a linear rowat the proximal portion 456 of the device 400. In some embodiments, thepitch 458 between adjacent fluid inlet ports is approximately 9 mm inorder to accommodate a standard 8-channel pipette.

In certain embodiments, the number of channels of the pipette matchesthe number of fluid inlet ports and the channel pitch matches the portpitch, such that there is a one-to-one correspondence between channelsof the multi-channel pipette and fluid inlet ports of the microfluidicdevice. In such embodiments, a single pipette may be used toconcurrently load samples into all of the fluid inlet ports at once,e.g., an 8-channel pipette having a 9 mm pitch is used to load samplesinto a device with 8 fluid inlet ports having a 9 mm pitch, a 12-channelpipette having a 4.5 mm pitch is used to load samples into a device with12 fluid inlet ports having a 4.5 mm pitch, etc.

In alternative embodiments, the number of channels of the pipettediffers from the number of fluid inlet ports, and/or the channel pitchdiffers from the port pitch, such that there is not a one-to-onecorrespondence between the pipette channels and the fluid inlet ports.

Optionally, the number of ports is a multiple of the number of channelsand/or the port pitch is a multiple of the channel pitch. In suchembodiments, multiple sequential loading steps can be used to loadsample into the fluid inlet ports, e.g., an 8-channel pipette having a 9mm pitch is used to load samples into a device with 16 fluid inlet portshaving a 4.5 mm pitch by first loading the odd ports, then the evenports (or vice-versa).

In some embodiments, a microfluidic device comprises a plurality offluid inlet ports with one or more of the following configurations: 2fluid inlet ports with a pitch of approximately 36 mm, 4 fluid inletports with a pitch of approximately 18 mm, 6 fluid inlet ports with apitch of approximately 12 mm, 8 fluid inlet ports with a pitch ofapproximately 9 mm, 12 fluid inlet ports with a pitch of approximately 9mm, 16 fluid inlet ports with a pitch of approximately 4.5 mm, 24 fluidinlet ports with a pitch of approximately 4.5 mm, 24 fluid inlet portswith a pitch of approximately 3 mm, 36 fluid inlet ports with a pitch ofapproximately 3 mm, 32 fluid inlet ports with a pitch of approximately2.25 mm, or 48 fluid inlet ports with a pitch of approximately 2.25 mm.

Materials and Compositions for Self-Digitization of Fluidic Samples

The microfluidic arrays and devices of the present disclosure can befabricated from a wide variety of materials. For instance, in someembodiments, the devices herein, or one or more components thereof,comprise a material selected from the following: polydimethylsiloxane(PDMS), polypropylene (PP), polychlorotrifluoroethylene (PCTFE),thermoset polyester (TPE), polymethylmethacrylate (PMMA), polyurethanemethacrylate, polyethylene, polyester (PET), polytetrafluoroethylene(PTFE), polycarbonate, parylene, polyvinyl chloride,fluoroethylpropylene, lexan, polystyrene, cyclic olefin polymers (COP),cyclic olefin copolymers, polyurethane, polyurethane blended withpolyacrylate, polyestercarbonate, polypropylene, polybutylene,polyacrylate, polycaprolactone, polyketone, polyphthalamide, celluloseacetate, polyacrylonitrile, polysulfone, an epoxy polymer, athermoplastic, a fluoropolymer, polyvinylidene fluoride, polyamide,polyimide, glass, quartz, silicon, a gallium arsenide, a siliconnitride, fused silica, ceramic, metal, or a combination thereof.Optionally, in various embodiments (e.g., for biological assays), adevice is fabricated from a polymer material so the device is disposablefor one-time use.

In some embodiments, the devices of the present disclosure comprise amaterial with suitable surface properties to facilitate the digitizationprocess. The surface properties of the devices or device components(e.g., channels and/or fluidic compartments) can be tailored for aspecific application. For example, some or all surfaces of the devicesare hydrophobic or hydrophilic in certain embodiments. In someembodiments, certain surfaces are hydrophobic and certain surfaces arehydrophilic. In some embodiments, the surfaces that are hydrophilic orhydrophobic are designed so as to allow loading of oils in certainchannels and/or fluidic compartments and aqueous solutions in certainchannels and/or fluidic compartments in the device.

For instance, in certain embodiments, one or more portions of amicrofluidic device (e.g., flow channel, fluidic compartments, devicebody, etc.) comprise a hydrophobic surface and/or are fabricated from ahydrophobic material, such as natively hydrophobic or surface-treatedpolydimethylsiloxane (PDMS), polycarbonate (PC), polypropylene (PP),glycol modified polyethylene terephthalate (PETG), cyclic olefincopolymer (COC), cyclic olefin polymer (COP),polychlorotrifluoroethylene (PCTFE), a multilaminate material, or acombination thereof.

In some embodiments, one or more portions of a microfluidic device(e.g., all of the flow channels and/or the plurality of fluidiccompartments) are modified with chemical and/or biological reagents torender the surfaces in contact with fluids preferential for wetting by aselected fluid (e.g., an oil). In certain embodiments, wetting primesthe surface of the device for discretization by facilitatingdisplacement of certain types of fluids from the device surfaces (e.g.,oils) and/or resisting displacement of certain types of fluids from thedevice surfaces (e.g., aqueous solutions).

A diverse variety of fluids or liquids can be used with the variousdevices, systems and methods of the present disclosure. In someembodiments, the fluids include water-based or aqueous solutions, forexample. In some embodiments, the fluids include liquids that aresparingly soluble in aqueous solutions, such as oils (e.g., fluorinatedoils, hydrocarbon oils, silicone oils, or mineral oils). Optionally,organic solvents are also used.

In certain embodiments, a fluid used with the devices, systems, andmethods herein comprises a fluidic sample (e.g., an aqueous solution)containing a variety of analytes, e.g., including but are not limitedto: chemicals, biochemicals, genetic materials (e.g., DNA, RNA, etc.),expressed products of genetic materials (e.g., proteins and/ormetabolites), crystallizing molecules, biological cells, exosomes,mitochondria, drugs, biological particles that circulate in peripheralblood or lymphatic systems, rare cells, particle, or combinationsthereof. Possible aqueous fluidic samples include but are not limitedto: various PCR and RT-PCR solutions, isothermal amplification solutionssuch as for LAMP or NASBA, blood samples, plasma samples, serum samples,solutions that contain cell lysates or secretions or bacterial lysatesor secretions, and other biological samples containing proteins,bacteria, viral particles and/or cells (eukaryotic, prokaryotic, orparticles thereof), among others. In certain embodiments, the fluidicsample also contains surfactants or other agents to facilitate desiredinteractions and/or compatibility with immiscible fluids (e.g., thefirst/third fluid) and/or the material of the device. In certainembodiments, the fluidic sample includes one or more of: cellsexpressing a malignant phenotype, fetal cells, circulating endothelialcells, tumor cells, cells infected with a virus, cells transfected witha gene of interest, or T-cells or B-cells present in the peripheralblood of subjects afflicted with autoimmune or autoreactive disorders,or other subtypes of immune cells, or rare cells or biological particles(e.g., exosomes, mitochondria) that circulate in peripheral blood or inthe lymphatic system or spinal fluids or other body fluids. The cells orbiological particles are optionally rare in a sample, such that thediscretization is used to spatially isolate the cells thereby allowingfor detection of the rare cells or biological particles, for example.

In some embodiments, the devices of the present disclosure function asat least a two-phase system, utilizing two or more immiscible fluids.For example, in some embodiments, a first fluid (e.g., an oil phase) isused to initially fill a device to displace any air. In someembodiments, the first fluid is configured to preferentially wet thedevice surface relative to a second fluid. Subsequently, in someembodiments, the second fluid, which is typically immiscible with thefirst fluid (e.g., an aqueous fluidic sample containing the sample ofinterest) is flowed through the device and enters the fluidiccompartments, displacing the oil. Optionally, a third fluid is thenflowed through the device to displace the aqueous phase within the mainflow channels but not the fluidic compartments. The third fluid istypically immiscible with the second fluid, and may or may not be thesame as the first fluid, and may or may not be miscible with the firstfluid. In certain embodiments, the fluidic compartments serve asshelters to isolate and digitize individual fluidic packets of theaqueous phase within the fluidic compartments. Optionally, the fluidiccompartments are substantially occupied by the aqueous phase such thatthe fluidic packets assume substantially the shape of the fluidiccompartment and the volume of the fluidic packet is substantiallydefined by the dimensions of the compartment. For example, if a fluidiccompartment is rectangular shaped, the fluidic packet contained within,can substantially assume a rectangular shape.

In certain embodiments, the first and/or third fluids each comprise anoil, such as a fluorinated oil, a hydrocarbon oil, a silicone oil, or acombination thereof. In some embodiments, the oil used as the firstand/or third fluid is a mineral oil-based oil, fluorocarbon-based oil,and/or silicone oil-based oil. Other embodiments can use other “oil”phases or alternative materials. In certain embodiments, the firstand/or third fluid also includes a surfactant and/or wetting agent toimprove desired interaction with the device surface and/or with thesecond fluid. In some embodiments, the first and the third fluid areidentical, while in other embodiments, the first and third fluid arecomposed of the same base material, but have differentsurfactant/additive concentrations and/or compositions. In yet otherembodiments, the third fluid is of a completely different compositionthan the first fluid and may or may not be miscible with the firstfluid. In certain embodiments, the first and/or third fluid containcomponents that interact with the second fluid and/or components withinthe second fluid. When a plurality of oils are used in a given method ofoperation (e.g., the first fluid, the third fluid, and/or the fourthfluid), the compositions of the oils can be the same or different. Insome embodiments, each of the oil compositions is independently selectedregardless of the composition of the other oils in use.

In some embodiments, the second fluid comprises a fluidic samplecontaining one or more analytes of interest. The second fluid isoptionally an aqueous solution. Examples of aqueous solutions includebut are not limited to: various PCR and RT-PCR solutions, isothermalamplification solutions such as for LAMP or NASBA, blood samples, plasmasamples, serum samples, solutions that contain cell lysates orsecretions or bacterial lysates or secretions, and other biologicalsamples containing proteins, bacteria, viral particles and/or cells(eukaryotic, prokaryotic, or particles thereof), or combinationsthereof.

In some embodiments, a fourth fluid is provided. In certain embodiments,the fourth fluid comprises an oil, such as a fluorinated oil, ahydrocarbon oil, a silicone oil, or a combination thereof. In someembodiments, the fourth fluid comprises a fluid that is compatible withan amplification reaction, such as PCR or isothermal amplification. Forinstance, in certain embodiments, the fourth fluid is used to displacethe third fluid in the flow channel before beginning an amplificationreaction to amplify a digitized analyte. The fourth fluid may bemiscible or immiscible with any of the first, second, and/or the thirdfluids. Optionally, in certain embodiments, the fourth fluid is the sameas the first fluid.

In some embodiments, a fifth fluid is provided. In certain embodiments,the fifth fluid comprises an oil, such as a fluorinated oil, ahydrocarbon oil, a silicone oil, or a combination thereof. In certainembodiments, the fifth fluid is used to flush the first, second, third,and/or fourth fluids from the device.

In some embodiments, any of the fluids herein (e.g., first, second,third, fourth, and/or fifth fluids) are provided to a fluid inlet porton the microfluidic device. The fluids may be provided to the same fluidinlet port or to different fluid inlet ports. In certain embodiments,the first, second, third, fourth, and/or fifth fluids discussed hereinare provided to a device in a sequential order that is different fromthe sequential orders provided herein. The first, second, third, fourth,and/or fifth fluids can be provided to a device in any order, and any ofthe first, second, third, fourth, and/or fifth fluids may be omitted insome embodiments of the present disclosure. The sequential ordersdescribed herein are exemplary and non-limiting to the practice of thedisclosed methods.

In various embodiments, the present disclosure provides methods forintroducing a fluid into a microfluidic device, the method comprising:providing a microfluidic device according to the present disclosure; andintroducing a first fluid into the flow channel of the microfluidicdevice. The terms “providing” and “introducing” are used interchangeablyherein to refer to the movement of fluid into or through a structure,such as for example an inlet or a channel.

In various embodiments, the present disclosure provides methods forintroducing a fluid into a microfluidic device, the method comprising:providing a microfluidic device according to the present disclosure; andintroducing a second fluid into the flow channel of the microfluidicdevice, wherein the second fluid is an aqueous solution.

In some embodiments, the second fluid comprises a fluidic samplecomprising an analyte and the method further comprises performing ananalysis of the analyte within at least one of the fluidic compartments.In certain embodiments, the analyte comprises a biological material. Infurther embodiments, the biological material is selected from a cell, abacteria, a virus, a prion, a nucleic acid, a protein, an expressedproduct of a genetic material, a crystallizing molecule, a particle, ora combination thereof. In yet further embodiments, the second fluidcomprises a first nucleic acid molecule and a second nucleic acidmolecule and the method further comprises distributing the first nucleicacid molecule into a first fluidic compartment, wherein the firstfluidic compartment does not comprise the second nucleic acid molecule.

In some embodiments, the present methods further comprise introducing afirst fluid into the flow channel of the microfluidic device, whereinthe first fluid is introduced into the flow channel before the secondfluid is introduced into the flow channel. In some embodiments, thefirst fluid comprises an oil. In further embodiments, the oil isselected from a fluorinated oil, a hydrocarbon oil, a silicone oil, or acombination thereof.

In some embodiments, the methods further comprise introducing a thirdfluid into the flow channel of the microfluidic device. In certainembodiments, the third fluid is introduced into the flow channel afterthe second fluid is introduced into the flow channel. In otherembodiments, the first fluid is an oil, the second fluid is an aqueoussolution, and the third fluid is an oil.

In some embodiments, the methods further comprise introducing a fourthfluid into the flow channel of the microfluidic device. In certainembodiments, the fourth fluid is introduced into the flow channel afterthe first fluid is introduced into the flow channel and before thesecond fluid is introduced into the flow channel. In other embodiments,the fourth fluid is introduced into the flow channel after the secondfluid is introduced into the flow channel and before the third fluid isintroduced into the flow channel. In some embodiments, the fourth fluidis introduced into the flow channel after the third fluid is introducedinto the flow channel. In further embodiments, the first fluid is anoil, the second fluid is an aqueous solution, the third fluid is an oil,the fourth fluid is an oil, and the fifth fluid is an oil, and whereinthe first, third, fourth, and fifth fluids are independently the same ordifferent from one another. In yet further embodiments, each of the oilsare independently selected from a fluorinated oil, a hydrocarbon oil, asilicone oil, or a combination thereof.

In some embodiments, the methods further comprise introducing a fifthfluid into the flow channel of the microfluidic device. In certainembodiments, the fifth fluid is introduced into the flow channel afterthe second fluid is introduced into the flow channel. In otherembodiments, the fifth fluid is introduced into the flow channel afterthe third fluid is introduced into the flow channel. In someembodiments, the method further comprises introducing a fourth fluidinto the flow channel, wherein the fourth fluid is introduced into theflow channel after the first fluid is introduced into the flow channeland before the second fluid is introduced into the flow channel. Incertain embodiments, the first fluid is an oil, the second fluid is anaqueous solution, the third fluid is an oil, the fourth fluid is an oil,and the fifth fluid is an oil, and wherein the first, third, fourth, andfifth fluids are independently the same or different from one another.In further embodiments, each of the oils are independently selected froma fluorinated oil, a hydrocarbon oil, a silicone oil, or a combinationthereof.

Systems for Self-Digitization, Processing, and Analysis of FluidicSamples

Various embodiments of the present disclosure provide systems forself-digitization, processing, and/or analysis of fluidic samples. Insome embodiments, a system is configured to discretize a fluidic sampleby driving fluid into the fluidic compartments of a microfluidic deviceby centrifugation. In certain embodiments, the system configured fordiscretizing a plurality of fluidic samples includes a rotor assemblyconfigured to receive one or more microfluidic devices, and a rotaryactuator configured to rotate the rotor assembly about an axis ofrotation (e.g., a central axis of the rotor assembly) in order togenerate centrifugal forces for driving fluid into the fluidiccompartments of the microfluidic device(s). The systems hereinadvantageously provide a simple and convenient format for simultaneousdiscretization of multiple fluidic samples suitable for high-throughputprocessing and analysis.

A rotor assembly for self-digitization of fluidic samples can beconfigured in a variety of ways. In some embodiments, a rotor assemblyincludes a central axis and a plurality of receptacles arranged radiallyaround the central axis. The rotor assembly can include any number ofreceptacles, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or morereceptacles. The distance between the proximal end of each of theplurality of receptacles and the central axis is within a range fromabout 10 mm to about 500 mm, from about 20 mm to about 300 mm, or fromabout 30 mm to about 200 mm, in some embodiments. The receptacles areeach shaped to receive one or more microfluidic devices of the presentdisclosure. For instance, in various embodiments, a receptacle is sizedand/or shaped to substantially match the size and/or shape of a devicebody of a microfluidic device (e.g., has a length of approximately 127.8mm and a width of approximately 85.5 mm, or a length of approximately100 mm and a width of approximately 75 mm). In certain embodiments, thereceptacles are shaped to removably receive and couple to themicrofluidic devices, e.g., via interference fits, snap fits, fasteners,latches, clamps, or other suitable coupling mechanisms.

In some embodiments, the receptacles are arranged such that the proximalbody portion of a received microfluidic device is positioned near thecentral axis of the rotor assembly, while the distal body portion of thedevice is positioned away from the central axis of the rotor assembly.Accordingly, in such embodiments, rotation of the rotor assembly causesfluid to be driven through the device from the proximal portion to thedistal portion. As discussed above and herein, the microfluidic deviceoptionally includes tapering flow channels with a decreasingcross-sectional dimension from the proximal portion to the distalportion in order to promote uniform fluid flow during centrifugation.

The rotary actuator can be any actuation mechanism suitable for rotatingthe rotor assembly about an axis of rotation, such as a brushless directcurrent motor, a brushed direct current motor, a servo motor, or astepper motor. In certain embodiments, the rotary actuator is configuredto rotate the rotor assembly at approximately 10 RPM, 50 RPM, 100 RPM,200 RPM, 300 RPM, 400 RPM, 500 RPM, 600 RPM, 700 RPM, 800 RPM, 900 RPM,1000 RPM, 1500 RPM, 2000 RPM, 2500 RPM, 3000 RPM, 3500 RPM, 4000 RPM,4500 RPM, or 5000 RPM. In various embodiments, the rotational speed ofthe rotary actuator is configured for driving fluid through themicrofluidic devices in order to discretize fluidic samples. Optionally,the rotational speed is configured for other applications, such as theimaging procedures discussed further herein. In certain embodiments, therotation of the rotary actuator is controlled by one or more processorsconfigured with instructions for controlling the operation of theself-digitization system, as discussed further herein.

FIG. 5 illustrates a rotor assembly 500 of a system forself-digitization of fluidic samples, in accordance with embodiments.The rotor assembly 500 includes a plurality of receptacles 502 (e.g., 12receptacles) arranged radially about a central axis 504. The centralaxis 504 is coupled to a rotary actuator 506, depicted herein as beingpositioned underneath the rotor assembly 500.

Each receptacle 502 is shaped to receive a corresponding microfluidicdevice 508. The microfluidic devices 508 can be similar to anyembodiment of the devices discussed herein, such as the device 300, 400,or 450. In certain embodiments, the devices 508 are positioned in theircorresponding receptacles 508 such that the proximal portion 510 of thedevice 508 is oriented towards the central axis 504 and the distalportion 512 of the device 508 is oriented away from the central axis504. As discussed above and herein, in some embodiments, the fluid inletports and fluid outlet ports of the device 508 are located at theproximal portion 510, the outlet reservoir is located at the distalportion 512, and the flow channels extend from the proximal portion 510to the distal portion 512.

In some embodiments, a rotor assembly is integrally formed as a singlepiece such that the receptacles cannot be decoupled from each otherwithout damaging the assembly. In such embodiments, the number ofreceptacles is fixed. In alternative embodiments, the rotor assemblyincludes removably coupled receptacles such that the number ofreceptacles can be adjusted according to user preference. Removablycoupled receptacles can be attached to the rotor assembly in a varietyof ways, including but not limited to fasteners (e.g., screws, pins),interlocking elements, snap fits, interference fits, latches, clamps,and the like.

FIGS. 6A and 6B illustrate a rotor assembly 600 with removably coupledreceptacles, in accordance with embodiments. FIG. 6A illustrates a firstconfiguration of the rotor assembly 600 in which a first removable rotorcomponent 602 is coupled to the central axis 604 of the assembly 600. Inthe depicted embodiment, the rotor component 602 includes a pair ofreceptacles 606 a, 606 b positioned opposite each other. Accordingly,the first configuration is suitable for spinning two microfluidicdevices simultaneously. FIG. 6B illustrates a second configuration ofthe rotor assembly 600 in which a first removable rotor component 602and a second removable rotor component 608 are coupled to the centralaxis 604, e.g., orthogonal to each other. The second removable rotorcomponent 608 is substantially similar to the first removable rotorcomponent 602. Accordingly, the second configuration is suitable forspinning four microfluidic devices simultaneously.

Various embodiments of the present disclosure provide systems anddevices configured to perform other functionalities in addition toself-digitization of fluidic samples via centrifugation.

In some embodiments, a system for self-digitization as discussed hereinalso incorporates components configured to perform one or more of thefollowing functions: heating of one or more fluidic samples in amicrofluidic device, cooling of one or more fluidic samples in amicrofluidic device, measuring a property of one or more fluidic samplesin a microfluidic device, and/or imaging one or more fluidic samples ina microfluidic device. For instance, various embodiments of the systemsherein are configured to perform discretization, thermal cycling, andimaging of a plurality of fluidic samples, e.g., for dPCR applications.Such multifunctional systems provide a convenient and compact unifiedplatform for high-throughput discretization, processing, and analysis offluidic samples.

For instance, in some embodiments, a multifunctional system for samplediscretization, processing, and analysis further includes one or moreheating devices configured to generate and apply heat to the fluidicsamples contained within the microfluidic devices received in the rotaryactuator. The heating devices can include a Peltier device, a convectionheater, radiation-emitting heater, a resistive heater, an inductiveheater, or a combination thereof. The heating devices can be configuredto apply heat with or without directly contacting the microfluidicdevices, as desired. In certain embodiments, application of heat to themicrofluidic devices is controlled by one or more processors configuredwith instructions for controlling the operation of the self-digitizationsystem, as discussed further herein.

As another example, in some embodiments, the systems herein furtherinclude one or more cooling devices for reducing the temperature of thefluidic samples contained within the microfluidic devices received inthe rotary actuator. The cooling devices can include a Peltier device, aconvection cooling device, or a combination thereof. The cooling devicescan be configured to apply cooling with or without directly contactingthe microfluidic devices, as desired. In certain embodiments,application of cooling to the microfluidic devices is controlled by oneor more processors configured with instructions for controlling theoperation of the self-digitization system, as discussed further herein.

FIGS. 7A through 7C illustrate exemplary rotor assemblies configured toaccommodate heating and/or cooling of microfluidic devices (e.g., viaconvective heating and/or cooling), in accordance with embodiments. FIG.7A illustrates a rotor assembly 700 with a plurality of receptacles 702(e.g., 12 receptacles) for receiving microfluidic devices (e.g., device704). Each receptacle 702 includes an opening shaped to receive thedevice and to expose the bottom surface of the device for thermalcoupling to a heating device and/or cooling device. In some embodiments,the rotor assembly includes an inner portion 706, middle portion 708,and outer portion 710 spanning the entire perimeter of the rotorassembly 700. The inner portion 706 and outer portion 710 are relativelythick, e.g., in order to provide structural support. Optionally, aclamping mechanism (not shown) is formed in the inner portion 706 and/orouter portion 710 in order to retain the device within the receptacle702. In certain embodiments, the middle portion 708 is relatively thin(e.g., has a thickness similar to the thickness of the device), e.g., inorder to facilitate air flow over and under the device for improved aircirculation and thermal transport.

FIG. 7B illustrates a rotor assembly 720 similar to the rotor assembly700, except that the outer portion 722 of the assembly 720 is formed asdiscrete sections rather than extending continuously around theperimeter. In some embodiments, the outer portion 722 includes aplurality of gaps 724 interspersed between the discrete sections suchthat the middle portion 726 extends to the edge of the rotor assembly720 at the gaps 724. This design provides additional improvements to aircirculation, in some embodiments. FIG. 7C illustrates a rotor assembly740 similar to the rotor assembly 720, except that additional openings742 are formed in the middle portion 744 of the assembly 740 to furtherimprove air flow. Optionally, the openings 742 are angled similar to fanblades in order to facilitate convective air flow and thermal transportwhen the rotor assembly 740 is spinning, e.g., during convective thermalcycling.

In certain embodiments, heating and/or cooling is applied to themicrofluidic devices received with the rotor assembly in accordance witha sequence or protocol, such as a dPCR thermal cycling procedure. Invarious embodiments, a thermal cycling procedure involves alternatinglyheating and cooling one or more fluidic samples (e.g., contained withinfluidic compartments of a microfluidic device) over a plurality ofcycles in order to amplify a target analyte (e.g., a nucleic acid). Theconditions for performing thermal cycling for analyte amplification areknown to those of ordinary skill in the art. Optionally, the applicationof heating and/or cooling is controlled by one or more processorsconfigured with suitable instructions for controlling the operation ofthe self-digitization system, as discussed further herein.

In order to improve the efficiency and uniformity of sample heatingand/or cooling, certain embodiments herein include a housing enclosingone or more components of the self-digitization system, such as therotor assembly, rotary actuator, heating device, and/or cooling device.Additionally, in some embodiments, the housing also protects thecomponents of the system from damage and/or contamination, and alsoprotects users from rapidly rotating parts and other potential safetyhazards. In various embodiments, one or more portions of the housing(e.g., at least one housing wall) are provided with insulatingstructures in order to reduce undesirable thermal conduction to or fromthe system. Optionally, one or more insulating structures are coupled tocomponents of the system that may be sensitive to temperaturefluctuations, excessively high temperatures, and/or excessive lowtemperatures, such as one or more portions of the rotary actuator (e.g.,a rotor shaft).

In some embodiments, the self-digitization system includes a ventilationassembly configured to control air flow around the heating device,cooling device, and/or rotor assembly, e.g., to produce more uniformheating and/or cooling of the microfluidic devices. Exemplaryventilation assembly components include one or more of fans, ducting,air inlets, air outlets, and the like.

FIG. 8 illustrates a portion of a system 800 configured for heatingand/or cooling of microfluidic devices, in accordance with embodiments.In some embodiments, the system 800 includes an insulated chamber 802enclosing the rotor assembly. The rotary actuator (e.g., motor 804) forrotating the rotor assembly optionally extends at least partiallyoutside of the chamber 802. In certain embodiments, the system 800includes a ventilation assembly for facilitating air flow and thermaltransport into and out of the chamber 802, such as one or more airintake vents 806 formed in the chamber 802, an air intake and heatingunit 808 coupled to the chamber 802 (e.g., via air intake duct 810), oneor more air exhaust vents 812 formed in the chamber 802, and/or an airexhaust unit 814 coupled to the chamber 802 (e.g., via air exhaust duct816). In some embodiments, the system 800 includes a heating deviceinside the chamber, rather than external to the chamber or incombination therewith. Optionally, the chamber 802 includes one or morewindows 818 that permit other functional components to access themicrofluidic devices from outside the chamber 802, such as an imagingdevice 820 as discussed further herein.

In some embodiments, the self-digitization system includes a heatingdevice composed of one or more devices which emit ultra violetradiation, emit visible radiation, emit infrared radiation, or emit acombination of two or more types of radiation. These radiation emittingheating devices will be referred to as lamps. Alternately or incombination, any combination of these lamps may be used. During heating,the lamps will be positioned to transfer heat to the microfluidicdevice. The lamps will irradiate the microfluidic device directly,irradiate a radiation absorbing material which then transfers heat tothe microfluidic device, or a combination thereof.

The lamps will be positioned below the rotor, above the rotor, alongsidethe rotor, or a combination thereof. The lamps will be aligned with themicrofluidic device by moving the microfluidic devices to be alignedwith the lamps, moving the lamps so that they are aligned with themicrofluidic devices, or a combination thereof. In some embodiments, theconfiguration and/or number of lamps renders this alignment stepunnecessary. In some embodiments, the lamps are attached to the rotor,attached inside the chamber but not directly to the rotor, or acombination thereof. In some embodiments, mirrors are used to directradiation from the lamps on to the microfluidic device, on to aradiation absorbing material which then transfers heat to themicrofluidic device, or a combination thereof.

Optionally, improved accuracy over the application of heating and/orcooling is achieved using feedback mechanisms, e.g., feedback based ontemperature data received from one or more temperature sensors. In someembodiments, one or more temperature sensors are used to monitor thetemperature of the microfluidic devices, rotor assembly, and/or interiorair temperature of the housing. In certain embodiments, the temperaturedata is received by one or more processors configured with instructionsto cause the system to adjust an amount of heating applied by theheating device and/or an amount of cooling applied by the cooling devicein response to the temperature data.

Various embodiments of the systems, methods, and devices herein aresuitable for obtaining measurements of the fluidic samples containedwithin the microfluidic devices. In some embodiments, measurement datais obtained after the fluidic samples have been discretized and/orprocessed (e.g., via heating, cooling, thermal cycling, etc.) todetermine the presence and/or amount of a target analyte present in thefluidic samples. For instance, in certain embodiments, the systemsherein are used to quantify an amount of a nucleic acid amplified viadPCR thermal cycling as discussed herein.

In some embodiments, the system includes an imaging device configured toobtain image data of the microfluidic devices received within the rotorassembly. In certain embodiments, the image data is bright-field imagedata, phase contrast image data, or fluorescence image data. In certainembodiments, the imaging modality is selected based on the properties ofthe target analyte in the fluidic sample, e.g., fluorescence imaging isused if the target analyte is fluorescent. Any imaging device withsufficient resolution for resolving the individual fluidic compartmentsof the device can be used with the systems, methods, and devices herein.For instance, in some embodiments, the imaging device is a microscope,such as a confocal microscope, spinning disk microscope, multi-photonmicroscope, planar illumination microscope, Bessel beam microscope,differential interference contrast microscope, phase contrastmicroscope, epifluorescent microscope, or a combination thereof. In someembodiments, a detector used in the systems, methods, and devices hereincan be a device capable of capturing an image such as a charged coupleddevice (CCD) camera or a complementary metal-oxide-semiconductor (CMOS)camera, a device with a single element sensitive to light such as anavalanche photo diode (APD) or photomultiplier tube (PMT), a hybriddevice such as a multielement APD or multielement PMT, or anycombination thereof. As another example, in some embodiments, theimaging device is a camera, such as a CCD camera or a CMOS camera. Forexample, the optics can comprise a single lens, a multielement lens, anoptical filter, a mirror, a beam splitter, or any combination thereof.Optionally, imaging of the microfluidic devices is controlled by one ormore processors configured with suitable instructions for controllingthe operation of the self-digitization system, as discussed furtherherein. In such embodiments, the imaging is configured to be controlledby a computer or other processing device via suitable software.

In certain embodiments, the imaging device is a digital single-lensreflex (DSLR) camera, e.g., with sufficient sensitivity and imageresolution for performing fluorescence imaging. In some embodiments, aDSLR camera-based imaging setup is advantageous in terms of beingrelatively low cost while providing satisfactory image resolution. Theconfiguration of the DSLR camera (e.g., ISO, F number, exposure time,focusing mode, etc.) can be varied as desired. In certain embodiments,the ISO, F number, and exposure time are fixed for a set of image datain order to permit comparison of signal intensities across differentimages. In some embodiments, the DSLR camera is used with a macro lens,e.g., having a lens mount matching the camera body.

In some embodiments, the imaging device is configured to obtain imagedata (e.g., fluorescence image data) of the microfluidic devices. Incertain embodiments, the field of view of the imaging device is sized tocapture a portion of a microfluidic device in a single image. Forinstance, in various embodiments, the imaging device comprises a fieldof view sized to capture at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 100% of a surface area of the body of a singlemicrofluidic device in a single image. In other embodiments, the fieldof view of the imaging device is sized to capture the entirety of asingle microfluidic device in a single image. In yet other embodiments,the field of view of the imaging device is sized to capture the entiretyof a plurality of microfluidic devices in a single image. Optionally,the field of view is adjustable, e.g., according to user preference. Invarious embodiments, the imaging device comprises a field of view sizedto capture at least 500, at least 1000, at least 1500, at least 2000, atleast 2500, at least 3000, at least 3500, at least 4000, at least 4500,at least 5000, at least 10,000, at least 50,000, or at least 100,000fluidic compartments of a single microfluidic device in a single image.

Subsequently, the image data is processed (e.g., with aid of one or moreprocessors of the system) in order to measure a characteristic (e.g.,fluorescence) from the fluidic compartments of the microfluidic devices.For instance, in some embodiments, the processing involves determining asignal intensity (e.g., fluorescence intensity) from each fluidiccompartment of the imaged microfluidic device in order to determine thepresence and/or concentration of a target analyte. A signal intensityabove a threshold value indicates presence of the analyte while a signalintensity below the threshold value indicates the analyte is absent, incertain embodiments. This approach is suitable for use with variousanalytical techniques, such as dPCR. Alternatively or in combination,the processing involves determining a signal wavelength (e.g.,fluorescence wavelength) from each fluidic compartment. This approach isadvantageous for multiplexed analysis, for example.

In certain embodiments, the imaging devices include and/or are used incombination with one or more illumination sources, such as anillumination source configured to provide substantially uniform floodingillumination, e.g., over the portion of the device to be imaged. Variousconfigurations of the illumination source and imaging device can beused. The optical axis of the imaging device and the principal rays ofthe illumination sources can form an angle between 0 and 10 degrees,between 10 and 20 degrees, between 20 and 30 degrees, between 30 and 45degrees, between 45 and 60 degrees, between 60 and 75 degrees, between75 and 90 degrees, between 90 and 105 degrees, between 105 and 120degrees, between 120 and 135 degrees, between 135 and 150 degrees,between 150 and 160 degrees, between 160 and 170 degrees, or between 170and 180 degrees. Alternately, or in combination, illumination sources atany combination of these angles are used. Angles greater than 90 degreesindicate that the illumination source(s) are on the opposite side of themicrofluidic device from the imaging device. When the angle between theoptical axis of the imaging device and the principal rays of theillumination source is between 0 and 10 degrees or between 170 and 180degrees, the illumination is referred to as direct illumination. Whenthe angle is between 10 and 170 degrees, the illumination is referred toas oblique. For instance, in some embodiments, direct illumination isused, e.g., in which the optical axis of the imaging device issubstantially parallel to the principal rays of the illuminationsource(s). Alternatively or in combination, oblique illumination isused, e.g., in which the optical axis of the imaging device is notparallel to the principal rays of the illumination source(s). In someembodiments, the illumination source includes one or more light-emittingdiodes (LEDs), such as a white light LED or colored LED, and the LEDscan be positioned relative to the imaging device in order to providedirect and/or oblique illumination, as desired. In some embodiments, theillumination source includes a ring illumination source positionedaround the imaging device and configured to provide direct and/oroblique illumination, as desired. In some embodiments, the illuminationcan be arranged in an epi-fluorescence configuration as the term isknown in the art, e.g., to provide direct illumination.

Optionally, in embodiments where the fluidic sample includes one or morefluorophores, the illumination source is configured to produce lightenergy at an excitation wavelength of the one or more fluorophores, andthe imaging device is configured to measure light energy at an emissionwavelength of the one or more fluorophores. The embodiments herein aresuitable for accommodating a wide variety of excitation wavelengths(e.g., within a range from approximately 350 nm to approximately 850 nm)and emission wavelengths (e.g., within a range from approximately 400 nmto approximately 900 nm). In certain embodiments, suitable excitationand/or emission filters are used to filter light transmitted to and/orreceived from the fluidic sample, respectively. Optionally, excitationfilters are not used if the illumination source is configured to producethe desired wavelength of light (e.g., a color LED).

FIG. 9A illustrates an oblique illumination configuration 900 forimaging a microfluidic device 902 using a DSLR camera 904, in accordancewith embodiments. It shall be appreciated that in alternativeembodiments, the configuration 900 can be adapted for use with othertypes of imaging devices. In some embodiments, the camera 904 includes amacro lens 906 and an emission filter 908 coupled to the lens 906 forreceiving light having a specified emission wavelength. One or moreillumination sources 910 (e.g., LED illumination sources) are positionedlaterally from the camera 904 and oriented towards the device 902 so asto form an illuminated region 912 over at least a portion of the device902. Optionally, the illumination sources 910 include an excitationfilter 914 for providing light having a specified excitation wavelength.In alternative embodiments, the excitation filter 914 is omitted, e.g.,when using a LED configured to emit light with the excitationwavelength. In certain embodiments, the illumination sources 910 areoriented at an angle relative to the optical axis of the camera 904 soas to provide oblique illumination of the device 902. In someembodiments, the illuminated region 912 covers the entirety of thedevice 902, while in other embodiments, the illuminated region 912covers only a portion of the device 902. In the latter case, the camera904 and/or illumination sources 910 are coupled to a movement stage orother actuation mechanism so as to sequentially illuminate and imagedifferent portions of the device 902. In some embodiments, theconfiguration 900 has identifiers that facilitate alignment of device904 or a portion thereof, e.g., a single well or multiple specificwells, for imaging with DSLR camera 902 or an alternative imagingdevice. Optionally, such identifiers are used to define illuminatedregion 912.

FIG. 9B illustrates a direct illumination configuration 950 for imaginga microfluidic device 952 using a DSLR camera 954, in accordance withembodiments. It shall be appreciated that in alternative embodiments,the configuration 950 can be adapted for use with other types of imagingdevices. In some embodiments, the camera 954 includes a macro lens 956.One or more illumination sources 958 are coupled to the camera 954(e.g., coupled to the macro lens 956) and oriented with a small angle tothe optical axis of the camera 954 so as to form an illuminated region960 over at least a portion of the device 952 and thereby provide directillumination of the device 952. In certain embodiments, the illuminationsource 958 is a ring illumination source (e.g., a LED ring light)positioned around at least a portion of the camera 954 (e.g., around themacro lens 956). Optionally, additional filters and/or other opticalcomponents are coupled to the camera 954, macro lens 956, and/orillumination source(s) 958, e.g., to provide light having a specifiedexcitation wavelength and/or receive light having a specified emissionwavelength. In some embodiments, the illuminated region 960 covers theentirety of the device 952, while in other embodiments, the illuminatedregion 960 covers only a portion of the device 952. In the latter case,the camera 954 and/or illumination source(s) 958 are coupled to amovement stage or other actuation mechanism so as to sequentiallyilluminate and image different portions of the device 952. In someembodiments, the configuration 950 has identifiers that facilitatealignment of device 952 or a portion thereof, e.g., a single well ormultiple specific wells, for imaging with DSLR camera 954 or analternative imaging device. Optionally, such identifiers are used todefine illuminated region 960. In some embodiments, the instrument canidentify, using the identifiers, the position and orientation of themicrofluidic chip relative to the imaging device thereby identifyingwhich portions of the image to analyze.

The various components (e.g., rotary actuator, heating device, coolingdevice, temperature sensor, ventilation assembly, imaging device,illumination source, etc.) of the self-digitization systems describedherein can be configured in multiple ways. In some embodiments, one ormore of the components herein are positioned above the rotor assemblyand received microfluidic devices. In some embodiments, one or more ofthe components herein are positioned below the rotor assembly andreceived microfluidic devices. In some embodiments, one or more of thecomponents herein are positioned on the rotor assembly, such as coupledto the rotor assembly.

In some embodiments, one or more of the components herein (e.g., rotaryactuator, heating device, cooling device, temperature sensor,ventilation assembly, imaging device, illumination source, etc.) aremovable, such that the position of the component(s) relative to therotor assembly is adjustable. For instance, in certain embodiments, acomponent is positioned near the rotor assembly when the component isoperating, and is positioned away from the rotor assembly when thecomponent is not operating. Optionally, the component is positioned nearthe rotor assembly when the rotor assembly is not rotating, and ispositioned away from the rotor assembly when the rotor assembly isrotating.

In some embodiments, an actuation mechanism (e.g., motor) is provided inorder to adjust the positions of one or more movable components relativeto the rotor assembly and/or microfluidic devices. Optionally, operationof the actuation mechanism is controlled by one or more processorsconfigured with suitable instructions for controlling the operation ofthe self-digitization system, as discussed further herein. In certainembodiments, the actuation mechanism is also configured to operablycouple one or more components (e.g., rotary actuator, heating device,cooling device, temperature sensor, ventilation assembly, imagingdevice, illumination source, a power source, etc.) to the rotorassembly. A component can be operably coupled to the rotor assembly withor without direct contact with the rotor assembly. For example, in someembodiments, a heating device is thermally coupled to the rotor assemblyin order to apply heat to the microfluidic devices via direct contactbetween the heating device and the rotor assembly and/or microfluidicdevices. In alternative embodiments, the heating devices are thermallycoupled to the rotor assembly without direct contact between the heatingdevice and the rotor assembly and/or microfluidic devices (e.g., thereis an air gap between the heating device and the rotor assembly and/ormicrofluidic devices).

In some embodiments, one or more heating devices are coupled to anactuation mechanism that translates and/or rotates the heating device(s)in order to thermally couple the heating device(s) with one or moremicrofluidic devices (or a portion thereof) to perform heating. Forinstance, in certain embodiments, the heating device(s) are positionedbelow the rotor assembly and the actuation mechanism raises the heatingdevice(s) in order to heat the microfluidic devices. Alternatively or incombination, the rotor assembly is moved (e.g., translated and/orrotated) in order to thermally couple one or more microfluidic deviceswith the heating device(s).

Similarly, in some embodiments, one or more cooling devices are coupledto an actuation mechanism that translates and/or rotates the coolingdevice(s) in order to thermally couple the cooling device(s) with one ormore microfluidic devices (or a portion thereof) to perform cooling. Forinstance, in certain embodiments, the cooling device(s) are positionedbelow the rotor assembly and the actuation mechanism raises the coolingdevice(s) in order to cool the microfluidic devices. Alternatively or incombination, the rotor assembly is moved (e.g., translated and/orrotated) in order to thermally couple one or more microfluidic deviceswith the cooling device(s).

As another example, in some embodiments, the imaging device is coupledto an actuation mechanism that translates and/or rotates the imagingdevice in order to align the imaging device (e.g., the field of view ofthe imaging device) with one or more microfluidic devices (or a portionthereof) to perform imaging. In embodiments where the field of view ofthe imaging device covers only a portion of the microfluidic device, theimaging device is moved to a plurality of different positions and/ororientations in order to allow for imaging of the entirety of themicrofluidic device. Alternatively or in combination, the rotor assemblyis moved (e.g., translated and/or rotated) in order to align one or moremicrofluidic devices with the imaging device.

FIG. 10 illustrates an exemplary multifunctional system 1000 for samplediscretization, heating, and imaging, in accordance with embodiments.The system 1000 can be used in combination with any embodiment of themethods and devices herein. The system 1000 includes a housing 1002 thatpartially or wholly encloses a rotor assembly 1004 for receiving aplurality of microfluidic devices. In some embodiments, the housing 1002includes a lid 1006 allowing access to the rotor assembly 1004 (e.g.,for loading and unloading microfluidic devices). The rotor assembly 1004is coupled to a rotary actuator (not shown) for rotating the rotorassembly 1004, e.g., in order to drive fluid into the fluidiccompartments of the received microfluidic devices to discretize asample. A plurality of heating devices (not shown) are positioned belowthe rotor assembly 1004 for heating the microfluidic devices, e.g., inaccordance with a thermal cycling procedure. In some embodiments, thenumber of heating devices corresponds to the number of receptacles inthe rotor assembly 1004, such that each heating device is used to applyheat to a single respective microfluidic device. The system 1000 alsoincludes an imaging device 1008 for imaging the microfluidic devices inthe rotor assembly 1004 and one or more illumination sources 1010 forilluminating the microfluidic devices during imaging. In someembodiments, the imaging device 1008 and illumination sources 1010 arepositioned above the rotor assembly 1004.

FIGS. 11A through 11C illustrate discretizing, heating, and imaging aplurality of fluidic samples using a multifunctional system, inaccordance with embodiments. The steps of the method can be performedusing any embodiment of the systems and devices herein. In someembodiments, the method is performed using a multifunctionalself-digitization system including a rotor assembly 1100 configured toreceive a plurality of microfluidic devices 1102, a rotary actuator 1104(e.g., a stepper motor, servo motor, brushless direct current motor,etc.) for rotating the rotor assembly 1100, a plurality of heatingdevices 1106 (e.g., Peltier units) coupled to an actuation mechanism1108 (e.g., a stage or other positional control system) and positionedbelow the rotor assembly 1100, and an imaging system positioned abovethe rotor assembly 1100 including an imaging device 1110 and at leastone illumination source 1112. Optionally, the system includes an encoderor other position information system for monitoring and/or controllingthe position of the rotor assembly 1100 relative to the heating devices1106 and/or the imaging system.

FIG. 11A illustrates sample discretization using the multifunctionalsystem. As discussed above and herein, sample discretization isperformed by rotating the microfluidic devices 1102 in the rotorassembly 1100 at a rotational speed sufficient to drive a fluidic sampleinto at least a subset of the fluidic compartments in the receivedmicrofluidic devices. The rotary actuator 1102 is used to rotate therotor assembly 1100 at a controlled speed. In some embodiments, theplurality of heating devices 1106 and actuation mechanism 1108 are in alowered position away from the rotor assembly 1100 during thediscretization step in order to allow for rotation of the rotor assembly1100.

FIG. 11B illustrates sample heating using the multifunctional system.The sample heating step is optionally performed after the samplediscretization step. In some embodiments, the actuation mechanism 1108is used to raise the heating devices 1106 towards the rotor assembly1100 in order to thermally couple the heating devices 1106 to themicrofluidic devices 1102. The thermal coupling may or may not involvedirect contact between the heating devices 1106 and the microfluidicdevices 1102. In some embodiments, the rotor assembly 1100 is stationaryduring the heating step. In alternative embodiments, such as whenconvective heating is used, the rotor assembly 1100 is rotating duringthe heating step, e.g., to improve air circulation for uniform heating.

FIG. 11C illustrates sample imaging using the multifunctional system.The sample imaging step is optionally performed after the sample heatingstep. In some embodiments, the rotor assembly 1100 is rotated by theactuator 1102 through a plurality of rotational positions in order tosequentially align each microfluidic device 1102 with the imaging device1110 and illumination source 1112 for imaging. In certain embodiments,the plurality of heating devices 1106 and actuation mechanism 1108 arein a lowered position away from the rotor assembly 1100 during theimaging step in order to accommodate rotation of the rotor assembly1100.

As discussed above and herein, the operation of the self-digitizationsystems herein can be controlled by one or more processors configuredwith suitable instructions in order to perform the various methodsherein. In some embodiments, for example, the systems described hereininclude a computer comprising one or more processors and a memory devicewith executable instructions stored thereon. In some embodiments, thecomputer is used to perform the methods described herein. In variousembodiments, a computer can be used to implement any of the systems ormethods illustrated and described above. In some embodiments, a computerincludes a processor that communicates with a number of peripheralsubsystems via a bus subsystem. These peripheral subsystems can includea storage subsystem, comprising a memory subsystem and a file storagesubsystem, user interface input devices, user interface output devices,and a network interface subsystem.

In some embodiments, a bus subsystem provides a mechanism for enablingthe various components and subsystems of the computer to communicatewith each other as intended. The bus subsystem can include a single busor multiple busses.

In some embodiments, a network interface subsystem provides an interfaceto other computers and networks. The network interface subsystem canserve as an interface for receiving data from and transmitting data toother systems from a computer. For example, a network interfacesubsystem can enable a computer to connect to the Internet andfacilitate communications using the Internet.

In some embodiments, the computer includes user interface input devicessuch as a keyboard, pointing devices such as a mouse, trackball,touchpad, or graphics tablet, a scanner, a barcode scanner, a touchscreen incorporated into the display, audio input devices such as voicerecognition systems, microphones, and other types of input devices. Ingeneral, use of the term “input device” is intended to include allpossible types of devices and mechanisms for inputting information to acomputer.

In some embodiments, the computer includes user interface output devicessuch as a display subsystem, a printer, a fax machine, or non-visualdisplays such as audio output devices, etc. The display subsystem can bea cathode ray tube (CRT), a flat-panel device such as a liquid crystaldisplay (LCD), or a projection device. Output devices are not limited toany particular device, and can include, for example, interactivedisplays, touchpads, touchscreens, as well as mobile devices such assmartphones and tablets. In general, use of the term “output device” isintended to include all possible types of devices and mechanisms foroutputting information from a computer.

In some embodiments, the computer includes a storage subsystem thatprovides a computer-readable storage medium for storing the basicprogramming and data constructs. In some embodiments, the storagesubsystem stores software (programs, code modules, instructions) thatwhen executed by a processor provides the functionality of the methodsand systems described herein. These software modules or instructions canbe executed by one or more processors. A storage subsystem can alsoprovide a repository for storing data used in accordance with thepresent disclosure. The storage subsystem can include a memory subsystemand a file/disk storage subsystem.

In some embodiments, the computer includes a memory subsystem that caninclude a number of memories including a main random access memory (RAM)for storage of instructions and data during program execution and a readonly memory (ROM) in which fixed instructions are stored. A file storagesubsystem provides a non-transitory persistent (non-volatile) storagefor program and data files, and can include a hard disk drive, a floppydisk drive along with associated removable media, a Compact Disk ReadOnly Memory (CD-ROM) drive, an optical drive, removable mediacartridges, and other like storage media.

The computer can be of various types including a personal computer, aportable computer, a workstation, a network computer, a mainframe, akiosk, a server or any other data processing system. Due to theever-changing nature of computers and networks, the description ofcomputer contained herein is intended only as a specific example forpurposes of illustrating the embodiments of the computer. Many otherconfigurations having more or fewer components than the system describedherein are possible.

Methods for Self-Digitization, Processing, and Analysis of FluidicSamples

Various methods for performing sample discretization, processing, andanalysis are suitable for use in combination with the embodimentsherein. In some embodiments, a method for discretizing and analyzing afluidic sample comprises: providing a microfluidic device as in any ofthe embodiments herein; applying a fluidic sample comprising a pluralityof discrete analytes to the fluid inlet ports of the microfluidicdevice; and rotating the microfluidic device such that the plurality ofdiscrete analytes are driven into a subset (e.g., less than all) of theplurality of fluidic compartments of the microfluidic device, therebydiscretizing the fluidic sample into a plurality of fluidic packetsdefined by the compartments. Optionally, the microfluidic device isconfigured for use with a multi-channel pipette and the fluidic sampleis applied to the fluid inlet ports using a multi-channel pipette.

In some embodiments, the discretization of the fluidic sample involvessequentially introducing a plurality of different fluids into themicrofluidic device. For example, in certain embodiments, the methodcomprises applying a first fluid to the microfluidic device (e.g., usinga multi-channel pipette), and rotating the microfluidic device to drivethe first fluid into the flow channels and/or fluidic compartments ofthe device. In various embodiments, the first fluid includes an oil thatis introduced into device, e.g., to displace any air bubbles. In certainembodiments, the method comprises applying a second fluid (e.g., anaqueous solution) comprising the fluidic sample into the microfluidicdevice via the fluid inlet ports, and rotating the microfluidic deviceto drive the second fluid into the fluidic compartments and displace thefirst fluid (e.g., the oil) from the fluidic compartments. After thesecond fluid is loaded, a third fluid (e.g., an oil that may or may notbe the same oil as the first fluid), is applied to the microfluidicdevice, and the device is rotated to displace the second fluid from thechannels, but not the fluidic compartments. As a result, the fluidicsample is compartmentalized into discrete volumes determined by thefluidic compartment dimensions.

As discussed above and herein, rotation of the microfluidic device canbe performed in various ways. In various embodiments, rotating themicrofluidic device comprises: providing a rotor assembly comprising acentral axis and a plurality of receptacles arranged radially around thecentral axis; positioning the microfluidic device in one of theplurality of receptacles such that the proximal body portion of themicrofluidic device is positioned near the central axis and the distalbody portion of the microfluidic device is positioned away from thecentral axis; and rotating the rotor assembly around the central axis,thereby rotating the microfluidic device.

In certain embodiments, the methods of the present disclosure are usedto discretize a fluidic sample including a plurality of nucleic acidmolecules, e.g., for PCR applications such as dPCR. In some embodiments,a fluid sample including a plurality of nucleic acid molecules isapplied to the fluid inlet ports of a microfluidic device. By rotatingthe microfluidic device as described herein, the nucleic acid moleculesare discretized within the fluidic compartments arranged in the devices,such that e.g., at least one nucleic acid molecule is present in atleast some of the fluidic compartments. In some embodiments, the nucleicacid molecules are discretized such that no more than one nucleic acidmolecule is present in at least some or all of the fluidic compartments.Alternatively, in some embodiments, the nucleic acid molecules arediscretized such that more than one nucleic acid molecule is present inat least some of the fluidic compartments. In some embodiments, thenucleic acid molecules are discretized such that at least one of thefluidic compartments does not include any nucleic acid molecules. Thenumber of nucleic acid molecules in each fluidic compartment depends insome embodiments on the concentration of the nucleic acid molecules inthe loaded fluidic sample and/or the size of the fluidic compartments.Optionally, reagents for performing PCR on the nucleic acid moleculesare also present in the sample that is loaded on the device. In someembodiments, after loading and discretization of the nucleic acidmolecules, PCR is conducted in situ in the microfluidic device, asdiscussed above and herein.

In some embodiments, the present disclosure provides methods forprocessing fluidic samples in a microfluidic device, subsequent tosample discretization. For instance, in certain embodiments, a methodcomprises applying heat to a microfluidic device in order to amplify aplurality of discrete analytes of a fluidic sample in the microfluidicdevice. As another example, in certain embodiments, a method comprisescooling the microfluidic device in order to reduce the temperature ofthe fluidic sample in the microfluidic device. In various embodiments,one or more heating and/or cooling cycles are performed in accordancewith a dPCR thermal cycling protocols. For example, in certainembodiments, the fluidic sample in the microfluidic device comprises aplurality of nucleic acids and one or more dPCR reagents, and theheating and/or cooling is applied according to a dPCR thermal cyclingprocedure in order to amplify the plurality of nucleic acids using theone or more dPCR reagents.

In some embodiments, the present disclosure provides methods foranalyzing fluidic samples in a microfluidic device, subsequent to samplediscretization and/or processing. For instance, in certain embodiments,a method comprises obtaining image data of the microfluidic device usingan imaging device. In various embodiments, image data is obtained byapplying light energy to the plurality of discrete analytes within theplurality of fluidic compartments of the microfluidic device, andmeasuring a fluorescence signal from the plurality of discrete analyteswithin the plurality of fluidic compartments. The imaging is optionallyperformed while the microfluidic devices are positioned in the rotorassembly. For instance, in certain embodiments, the microfluidic deviceis positioned in a receptacle of the rotor assembly, and image data isobtained by rotating the rotor assembly in order to align themicrofluidic device with the imaging device. Alternatively or incombination, the image data is obtained by translating and/or rotatingthe imaging device in order to align the microfluidic device with theimaging device.

In some embodiments, the methods of the present disclosure compriseproviding a device and/or system in accordance with any of theembodiments herein.

The specific dimensions of any of the apparatuses, devices, systems, andcomponents thereof, of the present disclosure can be readily varieddepending upon the intended application, as will be apparent to those ofskill in the art in view of the disclosure herein. Moreover, it isunderstood that the examples and embodiments described herein are forillustrative purposes only and that various modifications or changes inlight thereof may be suggested to persons skilled in the art and areincluded within the spirit and purview of this application and scope ofthe appended claims. Numerous different combinations of embodimentsdescribed herein are possible, and such combinations are considered partof the present disclosure.

As used herein A and/or B encompasses one or more of A or B, andcombinations thereof such as A and B.

All features discussed in connection with any embodiment or embodimentherein can be readily adapted for use in other embodiments andembodiments herein. The use of different terms or reference numerals forsimilar features in different embodiments does not necessarily implydifferences other than those expressly set forth. Accordingly, thepresent disclosure is intended to be described solely by reference tothe appended claims, and not limited to the embodiments disclosedherein.

Unless otherwise specified, the presently described methods andprocesses can be performed in any order. For example, a methoddescribing steps (a), (b), and (c) can be performed with step (a) first,followed by step (b), and then step (c). Or, the method can be performedin a different order such as, for example, with step (b) first followedby step (c) and then step (a). Furthermore, those steps can be performedsimultaneously or separately unless otherwise specified withparticularity.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentdisclosure only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual embodiments of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

While preferred embodiments of the present disclosure have been shownand described herein, it is to be understood that the disclosure is notlimited to the particular embodiments of the disclosure described, asvariations of the particular embodiments can be made and still fallwithin the scope of the appended claims. It is also to be understoodthat the terminology employed is for the purpose of describingparticular embodiments of the disclosure, and is not intended to belimiting. Instead, the scope of the present disclosure is established bythe appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure provided herein. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe disclosure provided herein.

All features discussed in connection with an embodiment or embodimentherein can be readily adapted for use in other embodiments andembodiments herein. The use of different terms or reference numerals forsimilar features in different embodiments does not necessarily implydifferences other than those expressly set forth. Accordingly, thepresent disclosure is intended to be described solely by reference tothe appended claims, and not limited to the embodiments disclosedherein.

EXAMPLES

The following examples are included to further describe some aspects ofthe present disclosure, and should not be used to limit the scope of theinvention.

Example 1 Imaging of a Microfluidic Device

This example describes an imaging procedure for obtaining image data ofa microfluidic device using a DSLR camera.

FIGS. 12A and 12B illustrate a configuration for imaging a microfluidicdevice. A DSLR camera 1200 with a macro lens 1202 is used. An emissionfilter 1204 is coupled to the front of the macro lens 1202. A pair ofLED illumination sources 1206 with an excitation filter 1208 are used toprovide oblique illumination of a microfluidic device 1210, thusproducing an illuminated region 1212 on the device 1210.

The DSLR camera is a Nikon D7100 (24.1 MP, 6000 by 4000 pixels). Thecamera configuration is manual mode, back button focus enabled, ISO 800,aperture F 4.0, and 8 second exposure time for fluorescence images. Thecamera is controlled from a computer using a Labview-based softwareprogram. A Tokina 100 mm f/2.8 macro lens is used.

The illumination (excitation) source is a white light LED with anunfiltered output of approximately 200 Lumens. The excitation filter hasa center wavelength of 475 nm with a bandwidth of 35 nm. The emissionfilter has a center wavelength of 520 nm with a bandwidth of 36 nm.

Images of the microfluidic device are obtained according the followingprocedure. First, the camera is turned on and connected to the computerin manual mode with back button focus enabled. Enabling back buttonfocus prior to image acquisition ensures that the camera does notattempt to autofocus before the fluorescent image is acquired. This issignificant as the fluorescence image is typically too dim for thecamera's autofocus mechanism to focus correctly.

The microfluidic device is illuminated with a white light LED withoutthe excitation filter. The software program controls the camera to focuson the device using autofocusing. Alternatively, the camera may bemanually focused to the correct location and locked in the desired focalposition. If desired, a white light image of the device is obtained. Thewhite light LED is then turned off, and a different white light LED withthe excitation filter is turned on. The exposure time is set to 8seconds and a fluorescence image is acquired. The resolution of thecamera is sufficient to image approximately one-fourth of the device ata time while maintaining adequate resolution to resolve individualcompartments in the device. By moving the device and/or camera, fourfluorescence images of the device are obtained and then analyzed.

FIGS. 13A through 13D illustrate exemplary imaging results obtained witha DSLR camera. FIG. 13A is an image of the illuminated region. FIG. 13Bis a graph illustrating the uniformity of the illumination over theilluminated region. FIG. 13C illustrates a fluorescence image obtainedby the camera. FIG. 13D is a graph illustrating uniformity of thefluorescence captured by the camera.

Example 2 Loading of Linear Centrifuge Device

This example describes loading of a linear centrifuge device. Theloading instrument included a stepper motor as the rotary actuator,capable of 1600 microsteps/rotation and >1200 rotations per minute.Attached to the rotary actuator was the rotor assembly containing tworeceptacles for securing the microfluidic devices. The rotary assemblyhad a housing that also anchored the rotary actuator to enable saferotation of the assembly and devices. The actuator was controlledthrough a computer on software provided by the manufacturer and/ordistributor of the rotary actuator. FIG. 14 provides an image of theloading instrument described in some embodiments of this example.

In this embodiment each device could contain up to 8 arrays, with eacharray comprised of 2560 fluidic compartments, and each compartmentcomprising a volume of approximately 23 nL (see FIG. 4B). The devices(FIGS. 3A and 3B) consisted of a 3″×4″ piece of glass that wasspin-coated with PDMS. Bonded to this was another piece of PDMScontaining the device features. This piece of PDMS consisted of threeregions. The central region is thin (between 300-1000 μm thick) andspanned the entire area encompassing the fluidic compartments. Theproximal region was thick (3-10 mm thick) and contained the inlets,inlet reservoirs, outlets and part of the channels that were in fluidiccommunication with the fluidic compartments. The distal region was alsothick (2-10 mm thick) and contained outlet reservoirs and portions ofthe channels that allowed for fluidic communication with the fluidiccompartments in the central region and the outlets in the proximalregion. Over the central region a vapor barrier was bonded. In someembodiments this was glass, in other embodiments it was PCTFE, in otherembodiments it was some other vapor barrier material. In someembodiments the vapor barrier was plasma sealed to the PDMS. In otherembodiments an adhesive layer was used.

In one embodiment the typical oil used to preload the device was 0.02%Abil WE 09, 33% Tegosoft DEC and 67% light mineral oil. The device wasdegassed in a vacuum desiccator then the oil was loaded into the inletreservoir and the device centrifuged in the rotor assembly at ˜900-1200RPM for 2-10 minutes. In some embodiments it was necessary to loadadditional oil into the inlet reservoir and additional rounds ofcentrifuging were performed. In some embodiments the partly loadeddevice was placed in the vacuum desiccator under low vacuum pressure tocontinue to evacuate air bubbles. Once all air bubbles were removed fromthe wells the device could be loaded with sample.

Sample loading occurred by adding sample (˜60-80 μL for this design) tothe inlet reservoir through the inlet with a pipette. In someembodiments the pipette was a multichannel pipette. Once sample wasloaded the device was loaded and secured onto the rotor and the rotorcentrifuged at 400-800 RPM for 4-10 minutes, or until all the sample hadsettled in the fluidic compartments or outlet reservoir. FIG. 15 is afluorescent image of a device loaded using this method. In thisembodiment six arrays were loaded with sample. The arrays containeddifferent sample solutions with different fluorescent intensities.

In particular arrays 2, 3 and 6 (counting from top to bottom) showgreater than 94% digitization of sample into fluidic compartments.

Example 3 Thermalcycling of Microfluidic Devices

This example describes one embodiment of thermalcycling to perform dPCRof the microfluidic device.

The device was loaded as described in example 2. After loading thedevice was imaged. In this embodiment the device was imaged as describedin example 1. In this embodiment the device was then placed on an insitu adapter of an Eppendorf Mastercycler thermalcycling instrument.Thermal paste had been put onto the adapter to improve the thermalcontact and improve the accuracy of the actual temperature of the deviceand the programmed temperature. A thin layer of mineral oil was used toimprove the thermal contact between the adapter and device byeliminating any air pockets that could be trapped between the devices.Thermalcylcing occurred based on the following program: 2 minutes at 95°C., then 35 cycles of 45 seconds at 95° C. and 60 seconds at 59° C.After thermalcycling the outside of the device was cleaned withisopropanol and the device was imaged again. FIG. 16 shows images of aregion of the device before (Top) and after (Bottom) Thermalcycling.Positive and negative wells can be clearly identified after performingdPCR, and the overall integrity of the array was well maintained.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A microfluidic array for discretizing a fluidicsample, the array comprising: a proximal array portion comprising afluid inlet port and a fluid outlet port; a distal array portion awayfrom the proximal portion; one or more flow channels each comprising alength extending from the proximal array portion to the distal arrayportion, a proximal end in fluidic communication with the fluid inletport, and a distal end in fluidic communication with the fluid outletport; and a plurality of fluidic compartments in fluidic communicationwith the one or more flow channels.
 2. The array of claim 1, wherein atleast one flow channel of the one or more flow channels comprises adecreasing cross-sectional dimension along the length from the proximalarray portion to the distal array portion.
 3. The array of claim 2,wherein the cross-sectional dimension of the at least one flow channeldecreases according to a tapering profile configured to producesubstantially uniform fluid flow rates along the length of the flowchannel.
 4. The array of any one of claims 2-3, wherein thecross-sectional dimension of the at least one flow channel decreasesaccording to a tapering profile configured to produce increasing flowresistance along the length of the flow channel.
 5. The array of any oneof claims 3-4, wherein the tapering profile comprises a continuoustapering profile.
 6. The array of claim 5, wherein the continuoustapering profile comprises a linear tapering profile, an exponentialtapering profile, a polynomial tapering profile, or a combinationthereof.
 7. The array of any one of claims 3-5, wherein the taperingprofile comprises a discontinuous tapering profile.
 8. The array ofclaim 7, wherein the discontinuous tapering profile comprises a steppedtapering profile.
 9. The array of any one of claims 2-8, wherein thecross-sectional dimension comprises a cross-sectional width.
 10. Thearray of claim 9, wherein the cross-sectional width comprises one ormore of an average cross-sectional width, a maximum cross-sectionalwidth, or a minimum cross-sectional width.
 11. The array of claim 9,wherein the cross-sectional width of the at least one flow channel isapproximately 80 μm near the proximal end and is approximately 50 μmnear the distal end.
 12. The array of claim 9, wherein thecross-sectional width of the at least one flow channel is within a rangefrom approximately 60 μm to approximately 120 μm near the proximal end,and is within a range from approximately 30 μm to approximately 70 μmnear the distal end.
 13. The array of claim 9, wherein thecross-sectional width of the at least one flow channel near the proximalend is approximately 1.2 times to approximately 2 times greater than thecross-sectional width near the distal end, or approximately 1 time toapproximately 3 times greater than the cross-sectional width near thedistal end.
 14. The array of claim 9, wherein the cross-sectional widthof the at least one flow channel decreases at an average rate within arange from approximately 0.2 μm/mm to approximately 0.75 μm/mm, fromapproximately 0.15 μm/mm to approximately 0.3 μm/mm, from approximately0.1 μm/mm to approximately 2 μm/mm, or from approximately 0.01 μm/mm toapproximately 10 μm/mm along the length of the flow channel.
 15. Thearray of any one of claims 2-8, wherein the cross-sectional dimensioncomprises a cross-sectional area.
 16. The array of claim 15, wherein thecross-sectional area of the at least one flow channel is approximately2400 μm² near the proximal end and is approximately 1500 μm² near thedistal end.
 17. The array of claim 15, wherein the cross-sectional areaof the at least one flow channel is a range from approximately 1200 μm²to approximately 4800 μm² near the proximal end, and is within a rangefrom approximately 600 μm² to approximately 2800 μm² near the distalend.
 18. The array of claim 1, wherein at least one flow channel of theone or more flow channels comprises a substantially constantcross-sectional dimension along the length from the proximal arrayportion to the distal array portion.
 19. The array of claim 1, whereinat least one flow channel of the one or more flow channels comprises anincreasing cross-sectional dimension along the length from the proximalarray portion to the distal array portion.
 20. The array of any one ofclaims 1-19, wherein each flow channel comprises a rectangular,trapezoidal, circular, semi-circular, oval, semi-oval, square, ortriangular cross-sectional shape.
 21. The array of any one of claims1-20, wherein the one or more flow channels comprise at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 16, at least 32, at least 64, at least128, at least 256, at least 512, at least 1024, at least 2048, at least4096, or at least 8192 flow channels.
 22. The array of any one of claims1-21, wherein the one or more flow channels are arranged to extendsubstantially parallel to each other.
 23. The array of any one of claims1-22, wherein the plurality of fluidic compartments comprises at least100, at least 500, at least 1000, at least 5000, at least 10,000, atleast 50,000, at least 100,000, at least 500,000 or at least 1 millionfluidic compartments.
 24. The array of any one of claims 1-23, whereineach fluidic compartment comprises a volume of approximately 5 pL, 10pL, 50 pL, 100 pL, 500 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, or 500 nL.25. The array of any one of claims 1-24, further comprising a fluidreservoir located at the proximal array portion, wherein the distal endof each flow channel is in fluidic communication with the fluid outletport via the fluid reservoir.
 26. The array of claim 25, wherein one ormore of the fluid inlet port, fluid outlet port, or the fluid reservoircomprises a depth of at least approximately 0.05 mm, 0.1 mm, 0.2 mm, 0.5mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or 6 mm.
 27. A microfluidic device fordiscretizing a fluidic sample, the device comprising: a body comprisinga proximal body portion and a distal body portion, and a plurality ofmicrofluidic arrays as in any one of claims 1-26 formed in the body suchthat the one or more flow channels of the plurality of microfluidicarrays extend substantially parallel to each other from the proximalbody portion to the distal body portion.
 28. The device of claim 27,wherein the body comprises a substantially rectangular shape.
 29. Thedevice of any one of claims 27-28, wherein the fluid inlet ports of theplurality of microfluidic arrays are arranged to receive a fluidicsample from a multi-channel pipette.
 30. The device of any one of claims27-29, wherein the fluid inlet ports of the plurality of microfluidicarrays are arranged in a row near the proximal body portion.
 31. Thedevice of claim 30, wherein the row comprises a pitch between adjacentfluid inlet ports of approximately 2.25 mm, 3 mm, 4.5 mm, 9 mm, 12 mm,18 mm, or 36 mm.
 32. The device of any one of claims 27-31, wherein theplurality of microfluidic arrays comprises 2, 4, 6, 8, 12, 16, 24, 32,36, or 48 microfluidic arrays.
 33. The device of any one of claims27-32, wherein the plurality of microfluidic arrays comprises at least500, at least 1000, at least 5000, at least 10,000, at least 50,000, atleast 100,000, at least 500,000, at least 1 million, at least 5 million,or at least 10 million fluidic compartments.
 34. The device of any oneof claims 27-33, wherein the body comprises a length of approximately127.8 mm and a width of approximately 85.5 mm.
 35. The device of any oneof claims 27-33, wherein the body comprises a length of approximately100 mm and a width of approximately 75 mm.
 36. The device of any one ofclaims 27-35, wherein the plurality of microfluidic arrays are formed inthe rectangular body by 3D printing, replica molding, injection molding,embossing, soft lithography, or a combination thereof.
 37. The device ofany one of claims 27-36, wherein the device is loaded with a firstfluid.
 38. A system for discretizing and analyzing fluidic samples, thesystem comprising: a rotor assembly comprising a central axis and aplurality of receptacles arranged radially around the central axis, eachreceptacle being shaped to receive a microfluidic device as in any oneof claims 27-36 such that the proximal body portion of the microfluidicdevice is positioned near the central axis and the distal body portionof the microfluidic device is positioned away from the central axis; arotary actuator coupled to the rotor assembly; and one or moreprocessors configured with instructions to cause the system to rotatethe rotor assembly around the central axis using the rotary actuator.39. The system of claim 38, wherein the rotor assembly comprises 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 receptacles.
 40. The system of any oneof claims 38-39, wherein a distance between a proximal end of each ofthe plurality of receptacles and the central axis is within a range fromabout 10 mm to about 500 mm, from about 20 mm to about 300 mm, or fromabout 30 mm to about 200 mm.
 41. The system of any one of claims 38-40,wherein the rotary actuator comprises a brushless direct current motor,a brushed direct current motor, or a stepper motor.
 42. The system ofany one of claims 38-41, wherein the rotary actuator is configured torotate the rotor assembly at approximately 10 RPM, 50 RPM, 100 RPM, 200RPM, 300 RPM, 400 RPM, 500 RPM, 600 RPM, 700 RPM, 800 RPM, 900 RPM, 1000RPM, 1500 RPM, 2000 RPM, 2500 RPM, 3000 RPM, 3500 RPM, 4000 RPM, 4500RPM, or 5000 RPM.
 43. The system of any one of claims 38-42, furthercomprising a heating device configured to generate heat, wherein the oneor more processors are configured with instructions to cause the systemto apply heat to the microfluidic devices received in the plurality ofreceptacles using the heating device.
 44. The system of claim 43,wherein the heating device comprises a Peltier device, a convectionheater, a radiation emitting heater, a resistive heater, an inductiveheater, or a combination thereof.
 45. The system of any one of claims43-44, wherein the heating device is configured to apply heat to themicrofluidic devices without directly contacting the microfluidicdevices.
 46. The system of any one of claims 43-45, wherein the one ormore processors are configured with instructions to cause the system toapply heat to the microfluidic devices according to a digital polymerasechain reaction (dPCR) thermal cycling procedure.
 47. The system of anyone of claims 43-46, further comprising a cooling device, wherein theone or more processors are configured with instructions to cause thesystem to reduce a temperature of the microfluidic devices received inthe plurality of receptacles using the cooling device.
 48. The system ofclaim 47, wherein the cooling device comprises a Peltier device, aconvection cooling device, or a combination thereof.
 49. The system ofany one of claims 43-48, further comprising a housing enclosing theheating device, rotor assembly, and rotor actuator.
 50. The system ofany one of claims 43-49, further comprising an actuation mechanismcoupled to the heating device, wherein the one or more processors areconfigured with instructions to cause the system to thermally couple theheating device with the microfluidic devices received in the pluralityof receptacles using the actuation mechanism.
 51. The system of any oneof claims 43-50, further comprising a ventilation assembly configured tocontrol air flow around the heating device and the rotor assembly inorder to produce uniform heating of the microfluidic devices.
 52. Thesystem of claim 51, wherein the ventilation assembly comprises one ormore of fans, ducting, air inlets, or air outlets.
 53. The system of anyone of claims 43-52, further comprising a temperature sensor configuredto obtain temperature data, wherein the one or more processors areconfigured with instructions to cause the system to adjust an amount ofheat applied by the heating device in response to the temperature data.54. The system of any one of claims 43-53, further comprising one ormore insulating structures coupled to: at least a portion of the rotorassembly; at least a portion of the rotary actuator, a rotor shaft ofthe rotary actuator; at least one wall of a housing enclosing the rotorassembly and the rotary actuator; or a combination thereof.
 55. Thesystem of any one of claims 38-54, wherein the one or more processorsare configured with instructions to cause the system to operativelycouple the rotor assembly to a power source, the rotary actuator, aheating device, a cooling device, a temperature sensor, or a combinationthereof.
 56. The system of claim 55, wherein the rotor assembly isoperatively coupled to the power source, the rotary actuator, theheating device, the cooling device, the temperature sensor, or acombination thereof without direct contact.
 57. The system of any one ofclaims 38-56, further comprising an imaging device configured to obtainimage data, wherein the one or more processors are configured withinstructions to cause the system to obtain image data of themicrofluidic devices received in the plurality of receptacles using theimaging device.
 58. The system of claim 57, wherein the imaging devicecomprises a camera.
 59. The system of claim 58, wherein the cameracomprises a digital single-lens reflex (DSLR) camera.
 60. The system ofany one of claims 57-59, wherein the imaging device is configured toobtain fluorescence image data of the microfluidic devices.
 61. Thesystem of claim 60, wherein the one or more processors are configuredwith instructions to cause the system to process the fluorescence imagedata in order to measure fluorescence from the pluralities of fluidiccompartments.
 62. The system of any one of claims 57-61, wherein theimaging device comprises a field of view sized to capture at least 500,at least 1000, at least 1500, at least 2000, at least 2500, at least3000, at least 3500, at least 4000, at least 4500, at least 5000, atleast 10,000, at least 50,000, or at least 100,000 fluidic compartmentsof a single microfluidic device in a single image.
 63. The system of anyone of claims 57-62, wherein the imaging device comprises a field ofview sized to capture at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or at least 100% of a surface area of the body of a singlemicrofluidic device in a single image.
 64. The system of any one ofclaims 57-63, wherein the one or more processors are configured withinstructions to cause the system to: rotate the rotor assembly using therotary actuator such that one of the microfluidic devices received inthe plurality of receptacles is aligned with the imaging device; andobtain image data of the microfluidic device using the imaging device.65. The system of any one of claims 57-64, wherein the one or moreprocessors are configured with instructions to cause the system to:translate or rotate the imaging device using an actuation mechanism suchthat a portion of one of the microfluidic devices received in theplurality of receptacles is aligned with the imaging device; and obtainimage data of the portion of the microfluidic device using the imagingdevice.
 66. The system of any one of claims 57-65, further comprising anillumination source configured to provide substantially uniform floodingillumination.
 67. The system of claim 66, wherein the illuminationsource is configured to provide oblique illumination.
 68. The system ofclaim 66, wherein the illumination source is configured to providedirect illumination.
 69. The system of any one of claims 66-68 whereinthe illumination source comprises one or more LEDs.
 70. The system ofany one of claims 66-69, wherein the illumination source comprises aring illumination source positioned around the imaging device.
 71. Thesystem of any one of claims 66-70, wherein the plurality of microfluidicdevices comprise a fluidic sample including a fluorophore, theillumination source is configured to produce light energy at anexcitation wavelength of the fluorophore, and the imaging device isconfigured to measure light energy at an emission wavelength of thefluorophore.
 72. The system of claim 71, wherein the excitationwavelength is within a range from approximately 350 nm to approximately850 nm.
 73. The system of any one of claims 71-72 wherein the emissionwavelength is within a range from approximately 400 nm to approximately900 nm.
 74. The system of any one of claims 38-73, further comprising aplurality of microfluidic devices as in any one of claims 27-36 receivedin the plurality of receptacles of the rotor assembly.
 75. A methodcomprising providing a device as in any one of claims 27-37.
 76. Amethod comprising providing a system as in any one of claims 38-74. 77.A method for discretizing and analyzing a fluidic sample, the methodcomprising: providing a microfluidic device as in any one of claims27-37; applying a fluidic sample to the fluid inlet ports of themicrofluidic device, the fluidic sample comprising a plurality ofdiscrete analytes; and rotating the microfluidic device such that theplurality of discrete analytes are driven into a subset of the pluralityof fluidic compartments of the microfluidic device.
 78. The method ofclaim 77, wherein the fluidic sample is applied to the fluid inlet portsusing a multi-channel pipette.
 79. The method of any one of claims77-78, wherein rotating the microfluidic device comprises: providing arotor assembly comprising a central axis and a plurality of receptaclesarranged radially around the central axis; positioning the microfluidicdevice in one of the plurality of receptacles such that the proximalbody portion of the microfluidic device is positioned near the centralaxis and the distal body portion of the microfluidic device ispositioned away from the central axis; and rotating the rotor assemblyaround the central axis, thereby rotating the microfluidic device. 80.The method of claim 79, wherein the rotor assembly is rotated atapproximately 10 RPM, 50 RPM, 100 RPM, 200 RPM, 300 RPM, 400 RPM, 500RPM, 600 RPM, 700 RPM, 800 RPM, 900 RPM, 1000 RPM, 1500 RPM, 2000 RPM,2500 RPM, 3000 RPM, 3500 RPM, 4000 RPM, 4500 RPM, or 5000 RPM.
 81. Themethod of any one of claims 77-80, wherein a distance between a proximalend of each of the plurality of receptacles and the central axis iswithin a range from about 10 mm to about 500 mm, from about 20 mm toabout 300 mm, or from about 30 mm to about 200 mm.
 82. The method of anyone of claims 77-81, further comprising applying heat to themicrofluidic device in order to amplify the plurality of discreteanalytes.
 83. The method of claim 82, wherein the fluidic samplecomprises a plurality of nucleic acids and one or more dPCR reagents,and wherein the heat is applied according to a dPCR thermal cyclingprocedure in order to amplify the plurality of nucleic acids using theone or more dPCR reagents.
 84. The method of any one of claims 82-83,wherein the heat is applied using a Peltier device, a convection heater,a radiation emitting heater, a resistive heater, an inductive heater, ora combination thereof.
 85. The method of any one of claims 77-84,further comprising cooling the microfluidic device using a Peltierdevice, a convection cooling device, or a combination thereof.
 86. Themethod of any one of claims 77-85, further comprising obtaining imagedata of the microfluidic device using an imaging device.
 87. The methodof claim 86, wherein obtaining the image data comprises: applying lightenergy to the plurality of discrete analytes within the plurality offluidic compartments; measuring a fluorescence signal from the pluralityof discrete analytes within the plurality of fluidic compartments. 88.The method of any one of claims 86-87, wherein the microfluidic deviceis positioned in a receptacle of a rotor assembly, and wherein obtainingthe image data comprises rotating the rotor assembly in order to alignthe microfluidic device with the imaging device.
 89. The method of anyone of claims 86-88, wherein obtaining the image comprises translatingor rotating the imaging device in order to align the microfluidic devicewith the imaging device.